High-affinity anti-human folate receptor beta antibodies and methods of use

ABSTRACT

Human anti-human folate receptor beta (FRβ) antibodies and antigen-binding fragments thereof are described, as well as methods of using such antibodies and fragments to treat a disorder, including but not limited to inflammatory disorders or cancers expressing cell surface FRβ.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional patent application of U.S. patentapplication Ser. No. 16/008,878 filed Jun. 14, 2018, which is anon-provisional application of and claims priority to U.S. ProvisionalPatent Application No. 62/519,387 filed Jun. 14, 2017, which isincorporated herein by reference in its entirety.

FIELD

The disclosure relates to folate receptor beta (FRβ) binding agents,such as FRβ antibodies, and more particularly, to human monoclonalantibodies, or antigen-binding fragments thereof, that specifically bindhuman FRβ and methods of using FRβ binding agents.

SUBMISSION OF SEQUENCE LISTING

The contents of the electronic submission of the text file SequenceListing, which is named MJL-39610-A.txt, which was created Jan. 8, 2019,and is 34.9 KB in size, is incorporated herein by reference in itsentirety.

BACKGROUND

Folic acid is a vitamin required for the synthesis of nucleotide basesand is essential for the proliferation of all cells. Folates also arerequired for production of S-adenosylmethionine, the common substrateused in methylation of DNA, histones, G proteins, and many metabolicbuilding blocks (see Kim, J Nutr 135:2703-2709 (2005); Loenen, BiochemSoc Trans 34:330-333 (2006)). Almost all cells take in folic acid viathe reduced folate carrier or proton coupled folate transporter (seeMatherly and Goldman, Vitam Horm 66:403-456 (2003)). Some cells,however, also express a folate receptor (FR) that binds folic acid about100,000 times tighter than the aforementioned transporters, and carriesbound folates into cells by receptor-mediated endocytosis (seeNakashima-Matsushita et al., Arth Rheum 42:1609-1616 (1999); Turk etal., Arthritis Rheumatoid 46:1947-1955 (2002)).

There are four members of the FR family: FRα, FRβ, FRγ and FR Δ (seeElnakat and Ratnam, Adv Drug Deliv Rev 56:1067-1084 (2004)). Differentisoforms of the FR are used by certain cancer cells, activatedmacrophages, and the proximal tubule cells of the kidney to capturefolates from their environment (see e.g., Nakashima-Matsushita et al.1999, supra; and Turk et al. 2002, supra). A need exists for reagentsand methods for differential targeting of the folate receptors fortreatment of disease.

BRIEF SUMMARY

The disclosure provides binding agents, such as antibodies, that bindFRβ proteins, as well as compositions, such as pharmaceuticalcompositions, comprising the binding agents. In certain embodiments, theFRβ-binding agents are novel polypeptides, such as antibodies, antibodyfragments, and other polypeptides related to such antibodies. In certainembodiments, the binding agents are antibodies that specifically bindhuman FRβ.

In one aspect, the disclosure provides a binding agent, such as anantibody, that specifically binds human FRβ. In some embodiments, theFRβ-binding agent or antibody modulates FRβ signaling, and/or inhibitsFRβ signaling, and/or inhibits activation of FRβ. In some embodiments,the FRβ-binding agent inhibits FRβ signaling. In some embodiments, theFRβ-binding agent inhibits or interferes with binding of folate to FRβ.

In certain embodiments, the FRβ-binding agent is an antibody. In otherembodiments, the antibody is a monoclonal antibody. In certainembodiments, the antibody is a humanized antibody. In certainembodiments, the antibody binds human FRβ. In certain embodiments, theFRβ-binding agent is the AS04498 antibody.

In certain embodiments, the antibody binds human FRβ with a K_(D) ofless than 10 nM. In certain embodiments, the antibody binds human FRβwith a K_(D) of less than 5 nM. In one embodiment, the antibody bindshuman FRβ with a K_(D) of less than 4 nM or with a K_(D) of less than2.5 nM. In one embodiment, the antibody binds human FRβ with a K_(D) ofless than 1 nM. In still another embodiment, the antibody binds humanFRβ with a K_(D) from 1 nM to 3 nM.

In one embodiment, the disclosure relates to methods of reducing thenumber of FRβ positive cells in a subject by administering a FRβ-bindingagent, including but not limited to an antibody. In one embodiment, thedisclosure relates to methods of reducing the number of activatedmacrophages expressing FRβ in a subject by administering a FRβ-bindingagent, including but not limited to an antibody.

In one embodiment, the disclosure further provides methods of inhibitingthe growth or survival of a cancer cell by administering the FRβ-bindingagents to a subject with a cancer. The disclosure further providesmethods of treating cancer by administering the FRβ-binding agents to asubject in need thereof. In some embodiments, the methods of treatingcancer or inhibiting tumor growth comprise targeting cancer cells withthe FRβ-binding agents. In certain embodiments, the methods comprisereducing the frequency of cancer cells in a tumor, reducing the numberof cancer cells in a tumor, reducing the tumorigenicity of a tumor,and/or reducing the tumorigenicity of a tumor by reducing the number orfrequency of cancer cells in the tumor.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a heavy chain Complementary Determining Region 1 (CDR1)comprising GYTFTYYA (SEQ ID NO:1), a heavy chain ComplementaryDetermining Region 2 (CDR2) comprising KYSQKFQ (SEQ ID NO:2), and aheavy chain Complementary Determining Region 3 (CDR3) comprisingARDISYGSFDYW (SEQ ID NO:3).

In some embodiments, the antibody further comprises a light chain CDR1comprising SLRSNY (SEQ ID NO: 4), a light chain CDR2 comprising GQF (SEQID NO:5), and a light chain CDR3 comprising DSRVSTGIHVVF (SEQ ID NO:6).

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises (a) a heavy chain CDR1 comprising GYTFTYYA (SEQ ID NO:1), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) aheavy chain CDR2 comprising KYSQKFQ (SEQ ID NO:2), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions and (c) a heavy chainCDR3 comprising ARDISYGSFDYW (SEQ ID NO:3), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions.

In some embodiments, the FRβ-binding agent is an antibody that comprises(a) a light chain CDR1 comprising SLRSNY (SEQ ID NO:4), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a lightchain CDR2 comprising GQF (SEQ ID NO:5), or a variant thereof comprising1, or 2 amino acid substitutions and (c) a light chain CDR3 comprisingDSRVSTGIHVVF (SEQ ID NO:6) or a variant thereof comprising 1, 2, 3, or 4amino acid substitutions.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises (a) a heavy chain CDR1 comprising GYTFTYYA (SEQ ID NO:1), or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) aheavy chain CDR2 comprising KYSQKFQ (SEQ ID NO:2), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; (c) a heavy chainCDR3 comprising ARDISYGSFDYW (SEQ ID NO:3), or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions; (d) a light chainCDR1 comprising SLRSNY (SEQ ID NO:4), or a variant thereof comprising 1,2, 3, or 4 amino acid substitutions; (e) a light chain CDR2 comprisingGQF (SEQ ID NO:5), or a variant thereof comprising 1, or 2 amino acidsubstitutions; and (f) a light chain CDR3 comprising DSRVSTGIHVVF (SEQID NO:6) or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions. In some embodiments, the amino acid substitutions areconservative amino acid substitutions.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises: (a) a heavy chain variable region having at least 80%sequence identity to SEQ ID NO:7; and/or (b) a light chain variableregion having at least 80% sequence identity to SEQ ID NO:11. In certainembodiments, the FRβ-binding agent is an antibody that comprises: (a) aheavy chain variable region having at least 90% sequence identity to SEQID NO:7; and/or (b) a light chain variable region having at least 90%sequence identity to SEQ ID NO:11.

In one embodiment, the FRβ-binding agent is an antibody that comprises aheavy chain region of SEQ ID NO: 8. In another embodiment, theFRβ-binding agent is an antibody that comprises a light chain region ofSEQ ID NO: 12. In still another embodiment, the FRβ-binding agent is anantibody that comprises a heavy chain region of SEQ ID NO: 8 and a lightchain region of SEQ ID NO: 12. In one embodiment, the FRβ-binding agentis AS04498 antibody.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a heavy chain region having at least 80% sequence identity toSEQ ID NO: 8 or a heavy chain region having at least 90% sequenceidentity to SEQ ID NO: 8. In certain embodiments, the FRβ-binding agentis an antibody that comprises a heavy chain region having at least 80%sequence identity to SEQ ID NO: 9 or a heavy chain region having atleast 90% sequence identity to SEQ ID NO: 9.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a light chain region having at least 80% sequence identity toSEQ ID NO: 12 or a light chain region having at least 90% sequenceidentity to SEQ ID NO: 12.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a heavy chain variable region of SEQ ID NO. 17. In anotherembodiment, the FRβ-binding agent is an antibody that comprises: (a) aheavy chain variable region having at least 80% sequence identity to SEQID NO:17; or a heavy chain variable region having at least 90% sequenceidentity to SEQ ID NO:17.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a heavy chain region having at least 80% sequence identity toSEQ ID NO: 18 or a heavy chain region having at least 90% sequenceidentity to SEQ ID NO: 18. In certain embodiments, the FRβ-binding agentis an antibody that comprises a heavy chain region having at least 80%sequence identity to SEQ ID NO: 19 or a heavy chain region having atleast 90% sequence identity to SEQ ID NO: 19.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a light chain variable region of SEQ ID NO. 15. In anotherembodiment, the FRβ-binding agent is an antibody that comprises: (a) alight chain variable region having at least 80% sequence identity to SEQID NO:15; or a light chain variable region having at least 90% sequenceidentity to SEQ ID NO:15.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a light chain region of SEQ ID NO. 16. In another embodiment,the FRβ-binding agent is an antibody that comprises a light chain regionhaving at least 80% sequence identity to SEQ ID NO: 16 or a light chainregion having at least 90% sequence identity to SEQ ID NO: 16.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a light chain variable region of SEQ ID NO. 20. In anotherembodiment, the FRβ-binding agent is an antibody that comprises: (a) alight chain variable region having at least 80% sequence identity to SEQID NO:20; or a light chain variable region having at least 90% sequenceidentity to SEQ ID NO:20.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a light chain region of SEQ ID NO. 21. In another embodiment,the FRβ-binding agent is an antibody that comprises a light chain regionhaving at least 80% sequence identity to SEQ ID NO: 21 or a light chainregion having at least 90% sequence identity to SEQ ID NO: 21.

In one embodiment, the disclosure relates to an antibody with a heavychain having a sequence selected from the group consisting of: SEQ IDNO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 17, SEQ ID NO. 18, and SEQID NO. 19, and a light chain having a sequence selected from the groupconsisting of: SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO.16, SEQ ID NO. 20, and SEQ ID NO. 21.

In one embodiment, the disclosure relates to an antibody with a heavychain having a sequence selected from the group consisting of: SEQ IDNO. 7, or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions, SEQ ID NO. 8, or a variant thereof comprising 1, 2, 3, or4 amino acid substitutions, SEQ ID NO. 9, or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions, SEQ ID NO. 17, or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions, SEQID NO. 18, or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions and SEQ ID NO. 19, or a variant thereof comprising 1, 2,3, or 4 amino acid substitutions and a light chain having a sequenceselected from the group consisting of: SEQ ID NO. 11, or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions, SEQ ID NO.12, or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions, SEQ ID NO. 15, or a variant thereof comprising 1, 2, 3,or 4 amino acid substitutions, SEQ ID NO. 16, or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions, SEQ ID NO. 20, or avariant thereof comprising 1, 2, 3, or 4 amino acid substitutions andSEQ ID NO. 21, or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions. In one embodiment, the variants referenced above can haveone or more amino acid substations in the variable region, the frameworkregion or both the variable region and the framework regions.

In another embodiment, the disclosure relates to a polypeptide selectedfrom the group consisting of: SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, and SEQ ID NO. 21.

In another embodiment, the disclosure relates to a compositioncomprising one or more polypeptides elected from the group consistingof: SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.12, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ IDNO. 19, SEQ ID NO. 20, and SEQ ID NO. 21.

In one embodiment, the disclosure relates to an antibody or antibodyfragment thereof having one or mutations in the V_(L) region as shown inTable 4, with sequence location corresponding to FIG. 2A and SEQ ID NO.24. In yet another embodiment, the disclosure relates to an antibody orantibody fragment thereof having one or mutations in the V_(L) region asshown in Table 4, with sequence location corresponding to FIG. 2B andSEQ ID NO. 25.

In another embodiment, the disclosure relates to compositions includingany of the FRβ-binding agents or antibodies described herein and apharmaceutically acceptable carrier, and methods of using suchcompositions. For example, a composition can be used in a method oftreating a patient having an inflammatory disorder (e.g.,atherosclerosis, ischemia/reperfusion injury, transplantation rejection,vasculitis, inflammatory osteoarthritis, glomerulonephritis, restenosis,systemic sclerosis, fibromyalgia, sarcoidosis, or an autoimmune diseasesuch as rheumatoid arthritis, systemic lupus erythematosus (SLE),ulcerative colitis, psoriasis, Type 1 diabetes, Crohn's disease,multiple sclerosis, and Sjogren's disease). The method can includeadministering to the patient an amount of a composition effective toreduce the number of FRβ positive macrophages and monocytes in thepatient.

In another embodiment, the disclosure relates to a method of depletingactivated macrophages from a human subject. The method includesadministering to the subject a human monoclonal antibody, orantigen-binding fragment thereof, that specifically binds human FRβ, inan amount effective to reduce the number of activated macrophages in thesubject.

In any of the methods described herein, the FRβ-binding agent orantibody can induce ADCC of FRβ expressing target cells.

In some embodiments, the antibody or fragment is de-fucosylated.

In any of the methods described herein, the FRβ-binding agent orantibody can induce opsonization-mediated clearance of FRβ expressingtarget cells.

In any of the methods described herein, the FRβ-binding agent orantibody can induce complement-mediated lysis of FRβ expressing targetcells.

In other aspects, the disclosure provides methods of inhibiting growthof a cancer cell, comprising contacting the cell or a bodily fluidcontaining the cell with an effective amount of a FRβ-binding agent orantibody, including each of those described herein.

In another aspect, the disclosure provides a method of inhibiting thegrowth of a cancer cell in a subject, comprising administering to thesubject a therapeutically effective amount of a FRβ-binding agent orantibody, including each of those described herein.

In another aspect, the disclosure provides a method of inhibiting folatereceptor signaling in a cell, comprising contacting the cell with aneffective amount of a FRβ-binding agent or antibody, including each ofthose described herein. In some embodiments, the cell is a tumor cell.In some embodiments, the tumor is a colorectal tumor. In someembodiments, the tumor is an ovarian tumor. In some embodiments, thetumor is a pancreatic tumor. In some embodiments, the tumor is a lungtumor. In some embodiments, the tumor expresses elevated levels of FRβ.In some embodiments, the tumor expresses elevated levels of FRβ. Incertain embodiments, the FRβ-binding agent inhibits growth of the tumor,for example, by reducing the number and/or frequency of cancer stemcells in the tumor.

In another aspect, the disclosure provides methods of treating cancer ina subject. In some embodiments, the method comprises administering to asubject a therapeutically effective amount of any of the FRβ-bindingagents or antibodies described above, as well as those describedelsewhere herein. In some embodiments, the cancer is acute myeloidleukemia. In some embodiments, the cancer is pancreatic cancer. In someembodiments, the cancer is colorectal cancer. In some embodiments, thecolorectal cancer comprises an inactivating mutation in the adenomatouspolyposis coli (APC) gene. In some embodiments, the colorectal cancerdoes not comprise an inactivating mutation in the APC gene. In someembodiments, the colorectal cancer comprises a wild-type APC gene. Insome embodiments, the cancer is ovarian cancer. In some embodiments, thecancer is breast cancer. In some embodiments, the cancer is lung cancer.In some embodiments, the cancer expresses elevated levels of FRβprotein. In some embodiments, the cancer is an ovarian cancer thatexpresses elevated levels of FRβ. In some embodiments, the cancer iscolon cancer that expresses elevated levels of FRβ. In some embodiments,the cancer is a pancreatic cancer that expresses elevated levels of FRβ.In some embodiments, the cancer is a breast cancer that expresseselevated levels of FRβ. In some embodiments, the cancer is a lung cancerthat expresses elevated levels of FRβ.

In another aspect, the disclosure provides methods of treating a diseasein a subject wherein the disease is associated with FRβ expression,and/or aberrant FRβ signaling comprising administering a therapeuticallyeffective amount of a FRβ-binding agent or antibody, including each ofthose described herein.

In certain embodiments of each of the aforementioned aspects, as well asother aspects and/or embodiments described elsewhere herein, thetreatment methods comprise administering a FRβ-binding agent incombination with at least one additional therapeutic agent. In someembodiments, the treatment methods comprise administering a FRβ-bindingagent in combination with a second FRβ-binding agent.

In certain embodiments of each of the aforementioned aspects, as well asother aspects and/or embodiments described elsewhere herein, thetreatment methods further comprise a step of determining the level ofFRβ protein expression in the tumor or cancer.

In another aspect, the disclosure provides a method of identifying ahuman subject or selecting a human subject for treatment with aFRβ-binding agent or antibody, including but not limited to, each ofthose described herein. In some embodiments, the method comprisesdetermining if the subject has a tumor that has an elevated expressionlevel of FRβ as compared to the expression of FRβ in normal tissue. Insome embodiments, the method comprises identifying a subject fortreatment or selecting a subject for treatment if the tumor has anelevated level of FRβ expression.

Pharmaceutical compositions comprising a FRβ-binding agent or antibodydescribed herein and a pharmaceutically acceptable carrier are furtherprovided, as are cell lines that produce the FRβ-binding agents. Methodsof treating cancer and/or inhibiting tumor growth in a subject (e.g., ahuman) comprising administering to the subject an effective amount of acomposition comprising the FRβ-binding agents are also provided.

Where aspects or embodiments of the disclosure are described in terms ofa Markush group or other grouping of alternatives, the disclosureencompasses not only the entire group listed as a whole, but also eachmember of the group individually and all possible subgroups of the maingroup, and also the main group absent one or more of the group members.The disclosure also envisages the explicit exclusion of one or more ofany of the group members in the claimed embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a depiction of the amino acid sequence of the full-length,unprocessed human FRβ protein (SEQ ID NO: 22). A fragment of thefull-length protein containing residues 22 to 236 was used to producerecombinant FRβ.

FIG. 2A is a depiction of the amino acid sequence of the m909 Fab heavychain sequence (SEQ ID NO: 24) and FIG. 2B is a depiction of the aminoacid sequence of the m909 Fab light chain lambda sequence (SEQ ID NO:25). Antibody m909 is a monoclonal antibody that binds human FRβ. Ineach of the sequences, the CDRs are underlined. By convention, numberingfor framework regions 1-4 (FR1-FR4) is consecutive from the N-terminus,with FR1 being the most N-terminal sequence (not shown). By convention,numbering for Complementary Determining Regions (CDRs 1-3) is alsoconsecutive from the N-terminus, with CDR1 being the most N-terminal(not shown).

FIG. 3A is a depiction of the amino acid sequence of the AS04498 Fabheavy chain sequence (SEQ ID NO: 8) and FIG. 3B is a depiction of theamino acid sequence of the AS04498 Fab light chain lambda sequence (SEQID NO: 12). In each of the sequences, the CDRs are underlined. Byconvention, numbering for framework regions 1-4 (FR1-FR4) is consecutivefrom the N-terminus, with FR1 being the most N-terminal sequence (notshown). By convention, numbering for Complementary Determining Regions(CDRs 1-3) is also consecutive from the N-terminus, with CDR1 being themost N-terminal (not shown).

FIG. 4A is a depiction of the amino acid sequence of the Fab heavy chain(SEQ ID NO: 8) of affinity matured antibody No. 2 binding to FRβ andFIG. 4B is a depiction of the amino acid sequence of the Fab light chainlambda sequence (SEQ ID NO: 16). In each of the sequences, the CDRs areunderlined. By convention, numbering for framework regions 1-4 (FR1-FR4)is consecutive from the N-terminus, with FR1 being the most N-terminalsequence (not shown). By convention, numbering for ComplementaryDetermining Regions (CDRs 1-3) is also consecutive from the N-terminus,with CDR1 being the most N-terminal (not shown).

FIG. 5A is a depiction of the amino acid sequence of the Fab heavy chain(SEQ ID NO: 18) of affinity matured antibody No. 3 binding to FRβ andFIG. 5B is a depiction of the amino acid sequence of the Fab light chainlambda sequence (SEQ ID NO: 21). In each of the sequences, the CDRs areunderlined. By convention, numbering for framework regions 1-4 (FR1-FR4)is consecutive from the N-terminus, with FR1 being the most N-terminalsequence (not shown). By convention, numbering for ComplementaryDetermining Regions (CDRs 1-3) is also consecutive from the N-terminus,with CDR1 being the most N-terminal (not shown).

FIG. 6A is a line graph displaying the binding properties of chimericIgG to FRβ at various concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25nM, 50 nM, 100 nM, 200 nM, 400 nM.

FIG. 6B is a line graph displaying the binding properties of m909 to FRβat various concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM,100 nM, 200 nM, 400 nM.

FIG. 6C is a line graph displaying the binding properties of AS04498 toFRβ at various concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50nM (×2), 100 nM, 200 nM, 400 nM.

FIG. 7 is a bar graph displaying the volume of a tumor in mice inrelation to the administration of the monoclonal antibody m909 or theaffinity matured (“AM”) AS04498 antibody. Both m909 and ASO4498 bindFRβ.

FIG. 8A is a flow cytometry graph of THP-1 cells, which express FRβ,displaying binding properties of the m909 antibody and the AS04498antibody at 4° C. at 0 hours.

FIG. 8B is a flow cytometry graph of THP-1 cells, which express FRβ,displaying binding properties of the m909 antibody and the AS04498antibody at 4° C. at 2 hours.

FIG. 8C is a flow cytometry graph of THP-1 cells, which express FRβ,displaying binding properties of the m909 antibody and the AS04498antibody at 4° C. at 4 hours.

FIG. 9A is a flow cytometry graph of THP-1 cells, which express FRβ,displaying binding properties of the m909 antibody and the AS04498antibody at 37° C. at 0 hours.

FIG. 9B is a flow cytometry graph of THP-1 cells, which express FRβ,displaying binding properties of the m909 antibody and the AS04498antibody at 37° C. at 2 hours.

FIG. 9C is a flow cytometry graph of THP-1 cells, which express FRβ,displaying binding properties of the m909 antibody and the AS04498antibody at 37° C. at 4 hours.

FIG. 10 is a bar graph displaying the binding affinity of the m909antibody and the AS04498 antibody in THP-1 cells expressing FRβ.

FIG. 11 is a bar graph displaying the efficacy of anti-FRβ antibodies inthe MV-411 xenograft AML Model.

FIG. 12A is a bar graph displaying depletion of mouse macrophages inblood samples using the M909 antibody and the AS04498 antibody.

FIG. 12B is a bar graph displaying depletion of mouse macrophages intumor samples using the M909 antibody and the AS04498 antibody.

FIG. 13 is a bar graph displaying the inhibition of CD68+, FRβ+ cellsusing m909 and AS04498 with the data presented as % control.

FIG. 14 is a bar graph displaying the inhibition of CD68+ cells usingm909 and AS04498 with the data presented as % control.

FIG. 15 is a bar graph displaying the inhibition of CD14+, FRβ+ cellsusing m909 and AS04498 with the data presented as % control.

FIG. 16 is a bar graph displaying the inhibition of CD14+ cells usingm909 and AS04498 with the data presented as % control.

FIG. 17 is a bar graph displaying the inhibition FRβ+ cells using m909and AS04498 with the data presented as % control.

DETAILED DESCRIPTION I. Definitions

To facilitate an understanding of the disclosure, a number of terms andphrases are defined below.

The terms “antagonist” and “antagonistic” as used herein refer to anymolecule that partially or fully blocks, inhibits, reduces orneutralizes a biological activity of a target and/or signaling pathway(e.g., FRβ binding or FRβ signaling). The term “antagonist” is usedherein to include any molecule that partially or fully blocks, inhibits,reduces or neutralizes the activity of a protein (e.g. FRβ protein).Suitable antagonist molecules specifically include, but are not limitedto, antagonist antibodies or antibody fragments.

The terms “modulation” and “modulate” as used herein refer to a changeor an alteration in a biological activity. Modulation includes, but isnot limited to, stimulating or inhibiting an activity. Modulation may bean increase or a decrease in activity (e.g., a decrease in FRβ binding;a decrease in FRβ signaling), a change in binding characteristics, orany other change in the biological, functional, or immunologicalproperties associated with the activity of a protein, pathway, or otherbiological point of interest.

The term “antibody” as used herein refers to an immunoglobulin moleculethat recognizes and specifically binds a target, such as a protein,polypeptide, peptide, carbohydrate, polynucleotide, lipid, orcombinations of the foregoing, through at least one antigen recognitionsite within the variable region of the immunoglobulin molecule. As usedherein, the term encompasses intact polyclonal antibodies, intactmonoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2,and Fv fragments), single chain Fv (scFv) antibodies, multispecificantibodies such as bispecific antibodies generated from at least twointact antibodies, monospecific antibodies, monovalent antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antigen determination portion of an antibody, andany other modified immunoglobulin molecule comprising an antigenrecognition site as long as the antibodies exhibit the desiredbiological activity. An antibody can be any of the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well-known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules, including but not limited to, toxins andradioisotopes.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, singlechain antibodies, and multispecific antibodies formed from antibodyfragments. “Antibody fragment” as used herein comprises anantigen-binding site or epitope binding site.

The term “variable region” of an antibody refers to the variable regionof the antibody light chain, or the variable region of the antibodyheavy chain, either alone or in combination. The variable regions of theheavy and light chain each consist of four framework regions (FR)connected by three complementarity determining regions (CDRs), alsoknown as “hypervariable regions.” The CDRs in each chain are heldtogether in close proximity by the framework regions and, with the CDRsfrom the other chain, contribute to the formation of the antigen-bindingsites of the antibody. There are at least two techniques for determiningCDRs: (1) an approach based on cross-species sequence variability (i.e.,Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5thEdition, National Institutes of Health, Bethesda Md.), and (2) anapproach based on crystallographic studies of antigen-antibody complexes(Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition,combinations of these two approaches are sometimes used in the art todetermine CDRs.

The term “monoclonal antibody” as used herein refers to a homogenousantibody population involved in the highly specific recognition andbinding of a single antigenic determinant or epitope. This is incontrast to polyclonal antibodies that typically include a mixture ofdifferent antibodies directed against different antigenic determinants.The term “monoclonal antibody” encompasses both intact and full-lengthmonoclonal antibodies as well as antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv), single chain (scFv) antibodies, fusion proteins comprisingan antibody portion, and any other modified immunoglobulin moleculecomprising an antigen recognition site (antigen-binding site).Furthermore, “monoclonal antibody” refers to such antibodies made by anynumber of techniques, including but not limited to, hybridomaproduction, phage selection, recombinant expression, and transgenicanimals.

The term “humanized antibody” as used herein refers to forms ofnon-human (e.g., murine) antibodies that are specific immunoglobulinchains, chimeric immunoglobulins, or fragments thereof that containminimal non-human sequences. Typically, humanized antibodies are humanimmunoglobulins in which residues of the CDRs are replaced by residuesfrom the CDRs of a non-human species (e.g., mouse, rat, rabbit, orhamster) that have the desired specificity, affinity, and/or bindingcapability (Jones et al., 1986, Nature, 321:522-525; Riechmann et al.,1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science,239:1534-1536).

In some instances, the Fv framework region residues of a humanimmunoglobulin are replaced with the corresponding residues in anantibody from a non-human species that has the desired specificity,affinity, and/or binding capability. The humanized antibody can befurther modified by the substitution of additional residues either inthe Fv framework region and/or within the replaced non-human residues torefine and optimize antibody specificity, affinity, and/or bindingcapability. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two or three, variabledomains containing all or substantially all of the CDRs that correspondto the non-human immunoglobulin whereas all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. The humanized antibody can also comprise at least a portion ofan immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin. Examples of methods used to generate humanizedantibodies are described in, for example, U.S. Pat. No. 5,225,539.

The term “human antibody” as used herein refers to an antibody producedby a human or an antibody having an amino acid sequence corresponding toan antibody produced by a human made using any of the techniques knownin the art. This definition of a human antibody specifically excludes ahumanized antibody comprising non-human antigen-binding residues.

The term “chimeric antibody” as used herein refers to an antibodywherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. Typically, the variable region of bothlight and heavy chains corresponds to the variable region of antibodiesderived from one species of mammals (e.g., mouse, rat, rabbit, etc.)with the desired specificity, affinity, and/or binding capability, whilethe constant regions are homologous to the sequences in antibodiesderived from another species (usually human) to avoid eliciting animmune response in that species.

The phrase “affinity matured antibody” as used herein refers to anantibody with one or more alterations in one or more CDRs thereof thatresult in an improvement in the affinity of the antibody for antigen,compared to a parent antibody that does not possess thosealterations(s).

The terms “epitope” and “antigenic determinant” are used interchangeablyherein and refer to that portion of an antigen capable of beingrecognized and specifically bound by a particular antibody. When theantigen is a polypeptide, epitopes can be formed both from contiguousamino acids and noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids (also referredto as linear epitopes) are typically retained upon protein denaturing,whereas epitopes formed by tertiary folding (also referred to asconformational epitopes) are typically lost upon protein denaturing. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation.

The terms “selectively binds” or “specifically binds” refers to abinding agent or an antibody that reacts or associates more frequently,more rapidly, with greater duration, with greater affinity, or with somecombination of the above to the epitope, protein or target molecule thanwith alternative substances, including unrelated proteins. In certainembodiments “specifically binds” means, for instance, that an antibodybinds a protein with a K_(D) of about 0.1 mM or less, but more usuallyless than about 1 μM. In certain embodiments, “specifically binds” meansthat an antibody binds a target at times with a K_(D) of at least about0.1 μM or less, at other times at least about 0.01 μM or less, and atother times at least about 1 nM or less. Because of the sequenceidentity between homologous proteins in different species, specificbinding can include an antibody that recognizes a protein in more thanone species (e.g., human FRβ and mouse FRβ). Likewise, because ofhomology within certain regions of polypeptide sequences of differentproteins, specific binding can include an antibody (or other polypeptideor binding agent) that recognizes more than one protein. It isunderstood that, in certain embodiments, an antibody or binding moietythat specifically binds a first target may or may not specifically binda second target. As such, “specific binding” does not necessarilyrequire (although it can include) exclusive binding, i.e. binding to asingle target. Thus, an antibody may, in certain embodiments,specifically bind more than one target. In certain embodiments, multipletargets may be bound by the same antigen-binding site on the antibody.For example, an antibody may, in certain instances, comprise twoidentical antigen-binding sites, each of which specifically binds thesame epitope on two or more proteins. In certain alternativeembodiments, an antibody may be bispecific or multispecific and compriseat least two antigen-binding sites with differing specificities. By wayof non-limiting example, a bispecific antibody may comprise oneantigen-binding site that recognizes an epitope on one protein (e.g.,human FRβ) and further comprise a second, different antigen-binding sitethat recognizes a different epitope on a second protein. Generally, butnot necessarily, reference to binding means specific binding

The terms “polypeptide” and “peptide” and “protein” are usedinterchangeably herein and refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids), as well as other modifications known in the art. It isunderstood that, because the polypeptides of this invention may be basedupon antibodies, in certain embodiments, the polypeptides can occur assingle chains or associated chains.

The terms “polynucleotide” and “nucleic acid” are used interchangeablyherein and refer to polymers of nucleotides of any length, and includeDNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a polymer by DNA or RNApolymerase.

“Conditions of high stringency” may be identified by those that: (1)employ low ionic strength and high temperature for washing, for example15 mM sodium chloride/1.5 mM sodium citrate/0.1% sodium dodecyl sulfateat 50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% FicolI/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75M NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC and50% formamide at 55° C., followed by a high-stringency wash consistingof 0.1×SSC containing EDTA at 55° C.

The terms “identical” or percent “identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are well-known in the art. These include,but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG WisconsinPackage, and variants thereof. In some embodiments, two nucleic acids orpolypeptides are substantially identical, meaning they have at least70%, at least 75%, at least 80%, at least 85%, at least 90%, and in someembodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acidresidue identity, when compared and aligned for maximum correspondence,as measured using a sequence comparison algorithm or by visualinspection. In some embodiments, identity exists over a region of thesequences that is at least about 10, at least about 20, at least about40-60 residues, at least about 60-80 residues in length or any integralvalue there between. In some embodiments, identity exists over a longerregion than 60-80 residues, such as at least about 80-100 residues, andin some embodiments the sequences are substantially identical over thefull length of the sequences being compared, such as the coding regionof a nucleotide sequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), β-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andantibodies disclosed herein do not abrogate the binding of thepolypeptide or antibody containing the amino acid sequence, to theantigen(s), i.e., FRβ to which the polypeptide or antibody binds.Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate antigen binding are well-known inthe art.

The term “vector” as used herein refers to a construct, which is capableof delivering, and usually expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid, or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, and DNA or RNA expressionvectors encapsulated in liposomes.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, a polypeptide, antibody, polynucleotide, vector, cell, orcomposition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material that isat least 50% pure (i.e., free from contaminants), at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure.

The terms “cancer” and “cancerous” as used herein refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. Examples of cancerinclude, but are not limited to, carcinoma, blastoma, sarcoma, andhematologic cancers such as lymphoma, leukemia and acute myeloidleukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass oftissue that results from excessive cell growth or proliferation, eitherbenign (noncancerous) or malignant (cancerous) including pre-cancerouslesions.

The term “metastasis” as used herein refers to the process by which acancer spreads or transfers from the site of origin to other regions ofthe body with the development of a similar cancerous lesion at the newlocation. A “metastatic” or “metastasizing” cell is one that losesadhesive contacts with neighboring cells and migrates via thebloodstream or lymph from the primary site of disease to invadeneighboring body structures.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumorinitiating cell” are used interchangeably herein and refer to cells froma cancer or tumor that: (1) have extensive proliferative capacity; (2)are capable of asymmetric cell division to generate one or more types ofdifferentiated cell progeny wherein the differentiated cells havereduced proliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Theseproperties confer on the cancer stem cells the ability to form orestablish a tumor or cancer upon serial transplantation into animmunocompromised host (e.g., a mouse) compared to the majority of tumorcells that fail to form tumors. Cancer stem cells undergo self-renewalversus differentiation in a chaotic manner to form tumors with abnormalcell types that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” refer to the total populationof cells derived from a cancer or tumor or pre-cancerous lesion,including both non-tumorigenic cells, which comprise the bulk of thecancer cell population, and tumorigenic stem cells (cancer stem cells).As used herein, the terms “cancer cell” or “tumor cell” will be modifiedby the term “non-tumorigenic” when referring solely to those cellslacking the capacity to renew and differentiate to distinguish thosetumor cells from cancer stem cells.

The term “tumorigenic” as used herein refers to the functional featuresof a cancer stem cell including the properties of self-renewal (givingrise to additional tumorigenic cancer stem cells) and proliferation togenerate all other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells).

The term “tumorigenicity” as used herein refers to the ability of arandom sample of cells from the tumor to form palpable tumors uponserial transplantation into immunocompromised hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, butnot limited to, humans, non-human primates, canines, felines, rodents,and the like, which is to be the recipient of a particular treatment.Typically, the terms “subject” and “patient” are used interchangeablyherein in reference to a human subject.

The term “pharmaceutically acceptable” refers to approved or approvableby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans.

The terms “pharmaceutically acceptable excipient, carrier or adjuvant”or “acceptable pharmaceutical carrier” refer to an excipient, carrier oradjuvant that can be administered to a subject, together with at leastone binding agent (e.g., an antibody) of the present disclosure, andwhich does not destroy the pharmacological activity thereof and isnontoxic when administered in doses sufficient to deliver a therapeuticeffect.

The terms “effective amount” or “therapeutically effective amount” or“therapeutic effect” refer to an amount of a binding agent, an antibody,polypeptide, polynucleotide, small organic molecule, or other drugeffective to “treat” a disease or disorder in a subject or mammal. Inthe case of cancer, the therapeutically effective amount of a drug(e.g., an antibody) has a therapeutic effect and as such can reduce thenumber of cancer cells; decrease tumorigenicity, tumorigenic frequencyor tumorigenic capacity; reduce the number or frequency of cancer stemcells; reduce the tumor size; reduce the cancer cell population; inhibitor stop cancer cell infiltration into peripheral organs including, forexample, the spread of cancer into soft tissue and bone; inhibit andstop tumor or cancer cell metastasis; inhibit and stop tumor or cancercell growth; relieve to some extent one or more of the symptomsassociated with the cancer; reduce morbidity and mortality; improvequality of life; or a combination of such effects. To the extent theagent, for example an antibody, prevents growth and/or kills existingcancer cells, it can be referred to as cytostatic and/or cytotoxic.

The terms “treating” or “treatment” or “to treat” or “alleviating” or“to alleviate” refer to: (1) therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of a diagnosed pathologiccondition or disorder and/or (2) prophylactic or preventative measuresthat prevent or slow the development of a targeted pathologic conditionor disorder. Thus, those in need of treatment include those already withthe disorder; those prone to have the disorder; and those in whom thedisorder is to be prevented.

In some embodiments, a subject is successfully “treated” according tothe methods disclosed herein if the patient shows one or more of thefollowing: a reduction in the number of or complete absence of cancercells; a reduction in the tumor size; inhibition of or an absence ofcancer cell infiltration into peripheral organs including the spread ofcancer cells into soft tissue and bone; inhibition of or an absence oftumor or cancer cell metastasis; inhibition or an absence of cancergrowth; relief of one or more symptoms associated with the specificcancer; reduced morbidity and mortality; improvement in quality of life;reduction in tumorigenicity; reduction in the number or frequency ofcancer stem cells; or some combination of effects.

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising” otherwise analogous embodiments described in termsof “consisting of” and/or “consisting essentially of” are also provided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

II. FRβ

In one embodiment, the disclosure provides a FRβ-binding agent orantibody as well as methods of using such FRβ-binding agent or antibodyto treat, detect, or monitor a cell expressing FRβ in a subject (e.g., ahuman patient). In one embodiment, the cell is a macrophage. In yetanother, the cell is an activated macrophage.

In one embodiment, the disclosure provides a FRβ-binding agent orantibody as well as methods of using such FRβ-binding agent or antibodyto treat, detect, or monitor an inflammatory disorder or a cancerexpressing FRβ in a subject (e.g., a human patient). The term “FRβ” asused herein refers to human folate receptor beta. The amino acidsequence of human FRβ can be found in FIG. 1 (SEQ ID NO: 22) and inGenBank under Accession No. NP_001107007. FRβ is a differential markeron the surface of myelomonocytic lineage cells. In normal tissues, FRβis expressed in placenta, myelomonocytic lineage cells (e.g., monocytesand macrophages), and mature neutrophils. FRβ, however, does not bindfolic acid on quiescent macrophages until the myeloid cell becomesactivated. FRβ is consistently detected in multiple myeloma cells,chronic myeloid leukemia (CIVIL) cells, and in 70% of acute myeloidleukemia (AML) cells. FRβ also has been detected by RT-PCR in solidtumors (e.g., carcinomas from colon, kidney, breast, ovary, uterus, orlung; squamous cell carcinomas of the head and neck; and malignancies ofnon-epithelial origin such as sarcomas, lymphomas, fibroushistiocytomas, ovarian granulosa cell tumor, astrocytoma, meningiomas,and Wilms' tumor). See, for example, Ross et al., Cancer, 73:2432-43(1994).

In monocytes and macrophage-lineage cells, FRβ expression is increasedupon activation. For example, FRβ is expressed and functional insynovial macrophages in rheumatoid arthritis patients. Furthermore,γ-scintigraphy images of patients with a variety of inflammatorydisorders (e.g., rheumatoid arthritis, Crohn's disease, ischemic boweldisease, Sjogren's syndrome, localized infections, atherosclerosis, andorgan transplant rejection) show uptake of a folate-targeted ^(99m)Tcimaging agent (EC20) at sites of inflammation (see Low et al., Acc ChemRes. 41(1):120-9 (2008); Matteson et al., Clin Exp Rheumatol 27:253-259(2009); and Ayala-Lopez et al., J Nuc Med, 51:768-774 (2010)). Thesefindings indicate expression of FRβ in activated macrophages andmonocytes.

The FRβ-binding agent or antibodies described herein specifically bindFRβ, and do not detectably bind FRα. In some embodiments, theFRβ-binding agents or antibodies described herein also do not detectablybind FRγ and/or FR-delta. FRα is expressed on the apical surfaces of afew epithelial cell types (primarily proximal tubules of the kidneys andalveolar epithelial cells of the lungs) and is upregulated on a varietyof epithelial-derived tumors. FRγ is rarely expressed and difficult todetect in vivo. FR-delta is expressed on regulatory T cells, where itexhibits only very low affinity for folic acid. As such, the FRβ-bindingagents or antibodies, which specifically bind FRβ, can be used fordepleting activated macrophages, treating inflammatory disorders, andtreating cancers that express FRβ.

III. FRβ-Binding Agents

The disclosure provides agents that bind human FRβ protein. These agentsare referred to herein as “FRβ-binding agents.” In some embodiments, theFRβ-binding agents are antibodies. In some embodiments, the FRβ-bindingagents are polypeptides. In certain embodiments, the FRβ-binding agentsbind FRβ.

In one embodiment, the FRβ-binding agent is a fully human, monoclonalantibody. In certain embodiments, the FRβ-binding agent is an antibodythat comprises a heavy chain CDR1 comprising GYTFTYYA (SEQ ID NO: 1), ora variant thereof comprising 1, 2, 3, or 4 amino acid substitutions. Inanother embodiment, the FRβ-binding agent is an antibody that comprisesa heavy chain CDR2 comprising KYSQKFQ (SEQ ID NO: 2), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions. In yetanother embodiment, the FRβ-binding agent is an antibody that comprisesa heavy chain CDR3 comprising ARDISYGSFDYW (SEQ ID NO: 3), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions.

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises a heavy chain CDR1 comprising GYTFTYYA (SEQ ID NO: 1), a heavychain CDR2 comprising KYSQKFQ (SEQ ID NO:2), and a heavy chain CDR3comprising ARDISYGSFDYW (SEQ ID NO: 3).

In certain embodiments, the FRβ-binding agent is an antibody thatcomprises: (a) a heavy chain CDR1 comprising GYTFTYYA (SEQ ID NO: 1), ora variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b)a heavy chain CDR2 comprising KYSQKFQ (SEQ ID NO: 2), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions and (c) aheavy chain CDR3 comprising ARDISYGSFDYW (SEQ ID NO: 3), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions.

In some embodiments, the FRβ-binding agent is an antibody that comprisesa light chain CDR1 comprising SLRSNY (SEQ ID NO: 4), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions. In someembodiments, the FRβ-binding agent is an antibody that comprises a lightchain CDR2 comprising GQF (SEQ ID NO: 5), or a variant thereofcomprising 1, or 2 amino acid substitutions. In some embodiments, theFRβ-binding agent is an antibody that comprises a light chain CDR3comprising DSRVSTGIHVVF (SEQ ID NO: 6) or a variant thereof comprising1, 2, 3, or 4 amino acid substitutions.

In some embodiments, the FRβ-binding agent is an antibody that comprisesa light chain CDR1 comprising SLRSNY (SEQ ID NO: 4), or a variantthereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a lightchain CDR2 comprising GQF (SEQ ID NO: 5), or a variant thereofcomprising 1, or 2 amino acid substitutions; and (c) a light chain CDR3comprising DSRVSTGIHVVF (SEQ ID NO: 6) or a variant thereof comprising1, 2, 3, or 4 amino acid substitutions.

In certain embodiments, the FRβ-binding agent is an antibody whichcomprises: an antibody that comprises (a) a heavy chain CDR1 comprisingGYTFTYYA (SEQ ID NO: 1), or a variant thereof comprising 1, 2, 3, or 4amino acid substitutions; (b) a heavy chain CDR2 comprising KYSQKFQ (SEQID NO: 2), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (c) a heavy chain CDR3 comprising ARDISYGSFDYW (SEQ IDNO: 3), or a variant thereof comprising 1, 2, 3, or 4 amino acidsubstitutions; (d) a light chain CDR1 comprising SLRSNY (SEQ ID NO: 4),or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;(e) a light chain CDR2 comprising GQF (SEQ ID NO: 5), or a variantthereof comprising 1, or 2 amino acid substitutions; and (f) a lightchain CDR3 comprising DSRVSTGIHVVF (SEQ ID NO: 6) or a variant thereofcomprising 1, 2, 3, or 4 amino acid substitutions. In some embodiments,the amino acid substitutions are conservative amino acid substitutions.

In some embodiments, the antibody or antigen binding fragment thereofcomprises the heavy chain variable region (V_(H)) amino acid sequenceset forth in SEQ ID NO: 7:EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMEIWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSF DYWGQGTLVTVSS.Alternatively, or in addition, the antibody or antigen binding fragmentthereof comprises the light chain variable region amino (Vi) amino acidsequence set forth in SEQ ID NO: 11:

SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGG TKLTVLG.

In some embodiments, an antibody or fragment thereof comprising suchsequences is designated AS04498 and specifically binds to human FRβ. Inone embodiment, AS04498 has a s heavy chain amino acid sequence as setforth in SEQ ID NO. 8 and a light chain amino acid sequence as set forthin SEQ ID NO. 12.

In some embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain amino acid sequence set forth in SEQ ID NO: 8:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH.

In some embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain amino acid sequence set forth in SEQ ID NO: 9:

MGWSWILLFLLSVTAGVHSEVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

In some embodiments, the antibody or antigen binding fragment thereofcomprises a light chain amino acid sequence set forth in SEQ ID NO: 12:

MGWSWILLFLLSVTAGVHSSSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

In some embodiments, the antibody or antigen binding fragment thereofcomprises the light chain variable region amino (Vi) amino acid sequenceset forth in SEQ ID NO:15:

SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGG TKLTVLG

In some embodiments, the antibody or antigen binding fragment thereofcomprises a light chain amino acid sequence set forth in SEQ ID NO: 16:

MGWSWILLFLLSVTAGVHSSSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

In some embodiments, the antibody or antigen binding fragment thereofcomprises the heavy chain variable region (V_(H)) amino acid sequenceset forth in SEQ ID NO:17:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGFTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISY GSFDYWGQGTLVTVSS.

In some embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain amino acid sequence set forth in SEQ ID NO 18:

EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGFTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH

In some embodiments, the antibody or antigen binding fragment thereofcomprises a heavy chain amino acid sequence set forth in SEQ ID NO 19:

MGWSWILLFLLSVTAGVHSEVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGFTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

In some embodiments, the antibody or antigen binding fragment thereofcomprises the light chain variable region amino (V_(l)) amino acidsequence set forth in SEQ ID NO: 20:

SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNLPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGG TKLTVLG

In some embodiments, the antibody or antigen binding fragment thereofcomprises a light chain amino acid sequence set forth in SEQ ID NO: 21:

MGWSWILLFLLSVTAGVHSSSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNLPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

It is understood that antibodies and antigen binding fragments describedabove, whether specified in terms of heavy and light chain components,or heavy or light chain components, can have any of the functionalproperties and activities described herein.

In some embodiments, the full length heavy or light chain, variableregion, CDR, framework region, or constant region of an antibody orantigen binding fragment described herein can have at least 80% sequenceidentity (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity) to the corresponding amino acid sequence set forth in any oneof SEQ ID Nos: 1-9, 11, 12, and 14-21. The percent identity between aparticular amino acid sequence and the amino acid sequence set forth inany one of SEQ ID Nos: 1-9, 11, 12, and 14-21 and can be determined asfollows. First, the amino acid sequences are aligned using the BLAST 2Sequences (Bl2seq) program from the stand-alone version of BLASTZcontaining BLASTP version 2.0.14. This stand-alone version of BLASTZ canbe obtained from the U.S. government's National Center for BiotechnologyInformation web site. Instructions explaining how to use the Bl2seqprogram can be found in the readme file accompanying BLASTZ. Bl2seqperforms a comparison between two amino acid sequences using the BLASTPalgorithm. To compare two amino acid sequences, the options of Bl2seqare set as follows: -i is set to a file containing the first amino acidsequence to be compared (e.g., C:\seq1.txt); -j is set to a filecontaining the second amino acid sequence to be compared (e.g.,C:\seq2.txt); -p is set to blastp; -o is set to any desired file name(e.g., C:\output.txt); and all other options are left at their defaultsetting. For example, the following command can be used to generate anoutput file containing a comparison will present those regions ofhomology as aligned sequences. If the two compared sequences do notshare homology, then the designated output file will not present alignedsequences. Similar procedures can be followed for nucleic acid sequencesexcept that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity is determined by dividing the number ofmatches by the length of the amino acid sequence in SEQ ID NOs: 1-9, 11,12, and 14-21, followed by multiplying the resulting value by 100.

It is noted that the percent identity value is rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2.It also is noted that the length value will always be an integer.

It will be appreciated that a number of nucleic acids can encode theamino acid sequences set forth in SEQ ID NOs: 1-9, 11, 12, and 14-21.The degeneracy of the genetic code is well known to the art; i.e., formany amino acids, there is more than one nucleotide triplet that servesas the codon for the amino acid. The nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 9 is set forth in SEQ ID NO: 10. Thenucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 isset forth in SEQ ID NO: 13. In some embodiments, a heavy or light chainof an antibody is encoded by a nucleic acid having at least 80% sequenceidentity (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequenceidentity) to the nucleotide sequences set forth in SEQ ID NOs.: 10 and13. Sequence identity is calculated as described above for proteinsequences except that blastn is used.

In one embodiment, an antibody or antigen birding fragment thereofdescribed herein can be de-fucosylated.

In some embodiments, an antibody or antigen binding fragment thereofdescribed herein induces antibody-dependent cell-mediated cytotoxicity(ADCC) of FRβ expressing target cells. Suitable antigen bindingfragments that induce ADCC have a functional Fc region, i.e., an Fcregion that can bind to an Fc receptor on an ADCC effector cell. Theantibodies and antigen binding fragments thereof can bind to IgG Fcbinding receptors (FcγR) receptors I, II and III (CD64, CD32, and CD16)in order to mediate the functional activities described herein. In someembodiments, binding to a FcγR (e.g., FcγR III) can be enhanced byremoving fucose residues from and/or by increasing galactosylation ofthe glycan present on the Fc portion of an IgG₁. See, for example,Houdes et al., Molecular & Cellular Proteomics 9:1716-1728 (2010);Kubota et al., Cancer Sci., 100: 1566-1572 (2009); Malphettes et al.,Biotechnol. Bioeng., 106:774-783 (2010); and Raju, Curr. Opin. Immunol.,20:471-478 (2008).

De-fucosylated antibodies can be produced using, for example, cells withreduced expression of the GDP-4,6-dehydratase gene (e.g., from amutation such as that in Chinese hamster ovary (CHO) Lec13 cells or froma small interfering RNA against the GDP-4,6-dehydratase gene), cells inwhich the .alpha.-1,6-fucosyltransferase (FUT8) has been knocked out orexpression reduced (e.g., using a small interfering RNA (siRNA) againstthe FUT8 gene), cells co-expressingβ-1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi amannosidase II (ManII), or cells expressingGDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD). See, Ishiguro et al.,Cancer Sci., pages 1-7, July, 2010; and von Horsten, Glycobiology,published online Jul. 23, 2010. FcγR binding also can be enhanced bymutating relevant amino acids in the heavy chain constant regionscomprising the Fc region (e.g., the hinge region, the C_(H)2 region, orthe C_(H)3 region). See, for example, Natsume et al., Drug Des. Develop.Ther., 3:7-16 (2009). In some embodiments, an antibody or fragmentthereof can induce opsonization-mediated clearance and/or inducecomplement-mediated lysis of FRβ expressing target cells. ADCC can beassessed in vitro using a lactate dehydrogenase (LDH) release assay orchromium-51 release assay.

In another embodiment, the disclosure relates to FRβ binding agents orantibodies that bind to particular epitopes of FRβ that contain, forexample, at least three amino acids of human FRβ (FIG. 1, SEQ ID NO:22). For example, an epitope can contain 3 to 30 such as 5 to 25, 7 to23, 10 to 20, or 13 to 18 amino acids of human FRβ). For example, anepitope can contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids ofhuman FRβ. The epitope can be in the N-terminal half (i.e., residues 1to 127 of SEQ ID NO: 9) or the C-terminal half of human FRβ (i.e.,residues 128 to 255 of SEQ ID NO:22).

In certain embodiments, a FRβ-binding agent or antibody described hereincan be conjugated or linked, either covalently or noncovalently, to avariety of molecules, including pharmaceutical agents, liposomes,oligonucleotides (e.g., small interfering RNA (siRNA)), toxins,detectable moieties, or biological molecules (e.g., a cytokine such asan interleukin (IL) 2, IL4, IL 12, 13 or 15; interferon (IFN), IFNα,IFNβ, or IFNγ) using methods known in the art. Conjugating or linkingsuch molecules to an antibody or antigen binding fragment describedherein allows the molecule to be targeted specifically to FRβ. As such,molecules can be delivered to the desired site while minimizingtoxicity.

For example, an antibody or antigen binding fragment can be conjugatedwith a pharmaceutical agent such as a chemotherapeutic (e.g., cisplatin,carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan,nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen,taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin,methotrexate, or an analog of any of the aforementioned). An antibody orantigen binding fragment also can be conjugated with ananti-inflammatory agent such as a glucocorticoid, nonsteroidalanti-inflammatory agent, phosphoinositide-3-kinase inhibitor (e.g.,wortmannin or derivatives such as demethoxyviridin, PX-866, LY294002,and LY294002 RGDS (Arg-Gly-Asp-Ser)-conjugated pro-drug SF1126, see,e.g., Maira et al., Biochem. Soc. Trans., 37, 265-272 (2009));NF-kappa-B inhibitor, I-kappa-B kinase inhibitor, mTOR (mammalian targetof rapamycin) inhibitor (e.g., rapamycin, CC1-779, RAD001, or AP23573,see Maira et al., supra), mitogen activated protein (MAP) kinaseinhibitor (e.g., SB-203580 and VX-745, see Brown et al., J.Inflammation, 5:22 (2008), or a Janus kinase (JAK) inhibitor.

In some embodiments, an antibody or antigen binding fragment also can beconjugated with a liposome. See, for example, the nanoliposomes of Lowet al. (Accounts of Chemical Research, 41(1):120-129 (2008)) that areless than 100 nm in diameter and contain a portion of PEGylated lipids(i.e., a lipid linked to polyethylene glycol (PEG)). Liposomes can beloaded with a pharmaceutical agent using methods known in the art.

In certain embodiments, an antibody or antigen binding fragmentdescribed herein can be linked to a toxin such as Pseudomonas exotoxin A(PE), diphtheria toxin (DT), gelonin, saporin, ricin A, abrin, mistletoelectin, modeccin, pokeweed antiviral protein (PAP), Bryodin 1, bouganin,or biologically active fragments thereof, to generate an immunotoxin.See, for example, Kreitman, BioDrugs, 23(1):1-13 (2009). PE and DT, andbiologically active fragments thereof, are particularly useful. Abiologically active fragment of PE can include, for example, amino acids253-364 and 381-613 of PE as described by Hassan et al., J Immunother.,23:473-9 (2000). A biologically active fragment of DT can include DT388or DAB389, which contain the first 388 or 389 amino acids of DT. See,for example, Chaudhary et al., Biochem Biophys Res Commun., 180:545-51(1991). Such immunotoxins are useful for killing FRβ expressing cells(e.g., any of the cancer cells described herein) in vivo or in vitro.

In certain embodiments, an antibody or antigen binding fragmentdescribed herein can be linked to a detectable moiety. Suitabledetectable moieties include, without limitation, radionuclides (e.g.,radionuclides used for in vivo diagnostics such as ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu,⁶⁷Cu, ²¹²Bi, ¹²³I, ¹³¹I, ²¹¹At, ¹⁷⁷Lu, ⁴⁷⁵c, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁵³Sm, ¹⁹⁹Au,^(99m)Tc, ¹¹¹In, ¹²⁴I, ¹⁸F, ¹¹C, ¹⁹⁸Au, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹³N, ^(34m)Cl,^(52m)Mn, ⁵⁵Co, ⁶²Cu, ⁶⁸Ga, ⁷²As, ⁷⁶As, ⁷²Se, ⁷³Se, or ⁷⁵Se; orradionuclides useful for in vitro experiments such as ¹²⁵I, ³⁵S, ³H,³²P, ³³P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, fluoresceinisothiocyanate (FITC), PerCP, rhodamine, or phycoerythrin (PE)),luminescent moieties (e.g., Qdot™ nanoparticles supplied by the QuantumDot Corporation, Palo Alto, Calif.), compounds that absorb light of adefined wavelength, or enzymes (e.g., alkaline phosphatase orhorseradish peroxidase). Antibodies can be indirectly labeled byconjugation with biotin then detected with avidin or streptavidinlabeled with a molecule described above. Methods of detecting orquantifying a detectable moiety depend on the nature of the moiety andare known in the art. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Methods of attaching the radionuclide atoms (or largermolecules/chelates containing them) to an antibody or antigen bindingfragment thereof are known in the art and can include incubating theantibody or fragment thereof with the radionuclide under conditions(e.g., pH, salt concentration, and/or temperature) which facilitatebinding of the radionuclide atom or radionuclide atom-containingmolecule or chelate to the antibody or antigen binding fragment (see,e.g., U.S. Pat. No. 6,001,329).

Other examples of detectable moieties that can be linked to an antibodyor antigen binding fragment include imaging agents, such as MRI orcomputed tomography (CT) contrast agents. Non-limiting examples of MRIcontrast agents include gadolinium and manganese chelates, iron salts,or gadolinium compounds such as gadodiamide (OMNISCA™), gadobenic acid(MULTIHANCE™), gadopentetic acid (MAGNAVIST™), gadoteridol (PROHANCE™)gadofosveset (ABLAVAR™), gadoversetamide (OPTIMARK™), or gadoxetic acid(EOVIST™, known). Non-limiting examples of CT contrast agents includeiodine based agents such as UROGRAFIN™, TELEBRIX™, GASTROGRAFIN™,OMNIPAQUE™, ULTRAVIST™, or VISIPAQUET™.

In one embodiment, the disclosure relates to a genetically engineeredconstruct comprising an antibody or antibody fragment thereof disclosedherein. In one embodiment, the disclosure relates to a geneticallyengineered construct comprising AS04498 or a fragment thereof.

In some embodiments, the disclosure relates to chimeric antigen receptorhaving an antibody or fragment thereof disclosed herein. Chimericantigen receptors, or CARs, are recombinant receptor constructs composedof an extracellular single-chain variable fragment (scFv) derived froman antibody, joined to a hinge/spacer peptide and a transmembranedomain, which is further linked to the intracellular T cell signalingdomains of the T cell receptor. CAR T cells combine the specificity ofan antibody with the cytotoxic and memory functions of T cells.

In some embodiments, a nucleic acid encoding an antibody or an antigenbinding fragment such as an scFv fragment is included in a construct forproducing a chimeric immune receptor. Chimeric immune receptorstypically include an extracellular portion and an intracellular portion,where the extracellular portion is an antigen binding fragment (e.g., ascFv fragment) having binding affinity for human FRβ and theintracellular portion is at least the intracellular domain of asignaling polypeptide such as the CD3 zeta chain, FcRy chain, or akinase such as a Syk cytoplasmic phosphotyrosine kinase. A chimericimmune receptor further can include at least the intracellular domain ofa costimulatory polypeptide such as the intracellular domain of CD28 orother costimulatory polypeptide such as OX40 (CD134), CD40L, PD-1, or4-1BB (CD137).

In some embodiments, a chimeric immune receptor includes an scFvfragment fused to the nonligand binding part of the extracellular andthe entire transmembrane and intracellular domains of CD28, which isfused with the intracellular domain of FcRγ. Constructs encodingchimeric immune receptors can be introduced ex vivo (e.g., using aretroviral vector) into T cells (e.g., cytotoxic T cells, CD4+ T cells,or CD8+ T cells) from peripheral lymphocytes of a given patient, and theresulting engineered T cells containing the chimeric receptor can bere-introduced into the patient. The engineered T cells can produce atleast one cytokine or lymphokine (e.g., IL2, IL3, IL4, IL5, IL6, IL9,IL10, IL12, or IFN-gamma.). Upon binding of the engineered T cells toFRβ expressing target cells, the engineered T cells are activated andcan kill the FRβ expressing target cells. See, for example, Eshhar in“Therapeutic Antibodies. Handbook of Experimental Pharmacology 181” Y.Chernajovsky, A. Nissim (eds.), 2008; and Pienert et al., Immunotherapy,1(6): 905-912 (2009).

In one embodiment, the disclosure relates to a CAR and the nucleic acidsequence encoding the CAR, wherein the CAR comprises an antigen bindingdomain, a transmembrane domain, a costinniiatory signaling region, and aCD3 zeta signaling domain, wherein the antigen binding domain is a FRbinding agent, An one embodiment, the FR-β binding agent is AS04498.

In certain embodiments, the FRβ-binding agent or antibody binds FRβprotein with a equilibrium dissociation constant (K_(D)) of about 1 μMor less, about 100 nM or less, about 40 nM or less, about 20 nM or less,about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. Insome embodiments, a FRβ-binding agent or antibody binds FRβ with a K_(D)of about 2.5 nM or less. In some embodiments, a FRβ-binding agent orantibody binds FRβ with a K_(D) of about 0.1 nM or less. In someembodiments, the dissociation constant of the binding agent (e.g., anantibody) to a FRβ protein is the dissociation constant determined usinga FRβ fusion protein comprising at least a portion of the FRβ proteinimmobilized on a Biacore chip.

In one embodiment, the FRβ-binding agent is AS04498 with a K_(D) of 2.5nM. The reported K_(D) for m909 is 57 nM.

In one embodiment, the FRβ-binding agent is an isolated, fully humanmonoclonal antibody that binds FRβ with a K_(D) of less than 2.5 nM.

In one embodiment, the FRβ-binding agent is an isolated, fully humanmonoclonal antibody that binds FRβ with a K_(D) from about 2.5 nM toabout 50 nM.

In certain embodiments, the FRβ-binding agent (e.g., an antibody) bindsto human FRβ protein with a half maximal effective concentration (EC₅₀)of about 1 μM or less, about 100 nM or less, about 40 nM or less, about20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nMor less.

In certain embodiments, the FRβ-binding agent is a recombinant antibody.In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is a chimeric antibody. In some embodiments,the antibody is a humanized antibody. In some embodiments, the antibodyis a fully human antibody. In some embodiments, the antibody is fullyhuman, man-made antibody.

In certain embodiments, the antibody is an IgG1 antibody. In certainembodiments, the antibody is an IgG2 antibody. In certain embodiments,the antibody is an antibody fragment comprising an antigen-binding site.In some embodiments, the antibody is monovalent, monospecific, bivalent,bispecific, or multispecific. In some embodiments, the antibody isconjugated to a cytotoxic moiety. In some embodiments, the antibody isisolated. In some embodiments, the antibody is substantially pure.

The FRβ-binding agents (e.g., antibodies) disclosed herein can beassayed for specific binding by any method known in the art. Theimmunoassays that can be used include, but are not limited to,competitive and non-competitive assay systems using techniques such asBiacore analysis, FACS analysis, immunofluorescence,immunocytochemistry, Western blots, radioimmunoassays, ELISA, “sandwich”immunoassays, immunoprecipitation assays, precipitation reactions, geldiffusion precipitin reactions, immunodiffusion assays, agglutinationassays, complement-fixation assays, immunoradiometric assays,fluorescent immunoassays, and protein A immunoassays. Such assays areroutine and well-known in the art (see, e.g., Ausubel et al., Editors,1994-present, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York, N.Y.).

For example, the specific binding of an antibody to human FRβ may bedetermined using ELISA. An ELISA assay comprises preparing antigen,coating wells of a 96 well microtiter plate with antigen, adding theFRβ-binding antibody or other FRβ-binding agent conjugated to adetectable compound such as an enzymatic substrate (e.g. horseradishperoxidase or alkaline phosphatase) to the well, incubating for a periodof time and detecting the presence of the antibody bound to the antigen.In some embodiments, the FRβ-binding antibody or agent is not conjugatedto a detectable compound, but instead a second conjugated antibody thatrecognizes the FRβ-binding antibody or agent is added to the well. Insome embodiments, instead of coating the well with the antigen, theFRβ-binding antibody or agent can be coated to the well and a secondantibody conjugated to a detectable compound can be added following theaddition of the antigen to the coated well. One of skill in the artwould be knowledgeable as to the parameters that can be modified toincrease the signal detected as well as other variations of ELISAs knownin the art.

In another example, the specific binding of an antibody to human FRβ maybe determined using FACS. A FACS screening assay may comprise generatinga cDNA construct that expresses an antigen as a fusion protein (e.g.,FRβ-Fc or FRβ-CD4TM), transfecting the construct into cells, expressingthe antigen on the surface of the cells, mixing the FRβ-binding antibodyor other FRβ-binding agent with the transfected cells, and incubatingfor a period of time. The cells bound by the FRβ-binding antibody orother FRβ-binding agent may be identified by using a secondary antibodyconjugated to a detectable compound (e.g., PE-conjugated anti-Fcantibody) and a flow cytometer. One of skill in the art would beknowledgeable as to the parameters that can be modified to optimize thesignal detected as well as other variations of FACS that may enhancescreening (e.g., screening for blocking antibodies).

The binding affinity of an antibody or other binding-agent to an antigen(e.g., a FRβ protein) and the off-rate of an antibody-antigeninteraction can be determined by competitive binding assays. One exampleof a competitive binding assay is a radioimmunoassay comprising theincubation of labeled antigen (e.g., ³H or ¹²⁵I), or fragment or variantthereof, with the antibody of interest in the presence of increasingamounts of unlabeled antigen followed by the detection of the antibodybound to the labeled antigen. The affinity of the antibody for anantigen (e.g., a FRβ protein) and the binding off-rates can bedetermined from the data by Scatchard plot analysis. In someembodiments, Biacore kinetic analysis is used to determine the bindingon and off rates of antibodies or agents that bind an antigen (e.g., aFRβ protein). Biacore kinetic analysis comprises analyzing the bindingand dissociation of antibodies from chips with immobilized antigen(e.g., a FRβ protein) on their surface.

IV. Methods of Using a FRβ Binding Agent or Antibody

In one embodiment, a FRβ binding agent, such as a human anti-human FRβmAb or antigen binding fragment thereof, is administered to a mammal,such as a human, with cells expressing FRβ. In one embodiment, a FRβbinding agent, such as a human anti-human FRβ mAb or antigen bindingfragment thereof, is administered to a mammal, such as a human, withmacrophages expressing FRβ. In one embodiment, a FRβ binding agent, suchas a human anti-human FRβ mAb or antigen binding fragment thereof, isadministered to a mammal, such as a human, with activated macrophagesexpressing FRβ.

In one embodiment, a FRβ binding agent, such as a human anti-human FRβmAb or antigen binding fragment thereof, is administered to a mammalsuch as a human that has been diagnosed with an inflammatory disorder ora cancer expressing cell surface FRβ. In some embodiments, engineered Tcells containing a chimeric immune receptor (see above section) areadministered to the human patient. Non-limiting examples of inflammatorydisorders include atherosclerosis, ischemia/reperfusion injury,transplantation rejection, vasculitis such as Wegener's granulomatosus,inflammatory osteoarthritis, glomerulonephritis, restenosis, systemicsclerosis, fibromyalgia, sarcoidosis, and autoimmune diseases.Non-limiting examples of autoimmune diseases include rheumatoidarthritis, systemic lupus erythematosus (SLE), ulcerative colitis,psoriasis, Type 1 diabetes (insulin-dependent diabetes mellitus),Crohn's disease, multiple sclerosis, and Sjogren's disease. Inflammatorydisorders also can include obstructive pulmonary diseases such as asthmaor chronic obstructive pulmonary disease (COPD), and IdiopathicPulmonary Fibrosis (IPF).

In one embodiment, a FRβ binding agent, such as a human anti-human FRβmAb or fragments described herein also can be administered to a subjectsuspected of having an inflammatory disorder. A subject “suspected ofhaving an inflammatory disorder” is one having one or more signs of thedisorder. Signs of such disorders are well-known to those of skill inthe art and include, without limitation, redness, swelling (e.g.,swollen joints), skin rashes, joint pain, joint pain, loss of jointfunction, fever, chills, fatigue, loss of energy, headaches, loss ofappetite, muscle stiffness, insomnia, itchiness, stuffy nose, sneezing,coughing, or one or more neurologic symptoms such as weakness,paresthesias, paralysis, dizziness, seizures, or pain. Signs of diabetesinclude, without limitation, higher than normal frequency of urination,unusual thirst, extreme hunger, unusual weight loss, extreme fatigue,visual problems, and irritability.

Non-limiting examples of cancers expressing cell surface FRβ includemyeloid cancers such as acute myeloid leukemia (AML) or chronic myeloidleukemia (CML), multiple myeloma, or a solid cancer containing FRβexpressing cells such as squamous cell carcinoma of the head and neck,or a malignancy of non-epithelial origin. Treatment of an inflammatorydisorder or cancer can include reducing the severity of the disorder orslowing progression of the disorder.

In one embodiment, a FRβ binding agent, such as a human anti-human FRβmAb or antigen binding fragment thereof, also can be administeredprophylactically in subjects at risk for developing an inflammatorydisorder to prevent development of symptoms of the disorder fromoccurring, delay onset of symptoms, or lessen the severity ofsubsequently developed disorder symptoms. A subject “at risk ofdeveloping an inflammatory disorder” refers to a subject with a familyhistory of one or more inflammatory disorders (e.g., a geneticpredisposition to one or more inflammatory disorders) or one exposed toone or more inflammation-inducing conditions.

For example, a subject can have been exposed to a viral or bacterialsuperantigen such as, but not limited to, Staphylococcal enterotoxins(SEs), a Streptococcus pyogenes exotoxin (SPE), a Staphylococcus aureustoxic shock-syndrome toxin (TSST-1), a Streptococcal mitogenic exotoxin(SME) and a Streptococcal superantigen (SSA). A FRβ binding agent, suchas a human anti-human FRPmAb or fragment thereof also can beadministered to deplete activated macrophages from a human subject. Inany case, an amount of a FRβ binding agent, such as a human anti-humanFRPmAb or antigen binding fragment thereof effective to reduce thenumber of FRβ positive cells (e.g., macrophages and monocytes, or cancercells) in the patient is administered. The number of FRβ positive cellscan be determined by doing cell counts or using a folate-targetedimaging agent.

Methods described herein can include, for example, monitoring thepatient to determine if the disorder is improving with treatment. Anymethod can be used to monitor an inflammatory disorder or cancer. Forexample, for rheumatoid arthritis patients, joint pain and/or stiffness,or bone erosion can be monitored in the patient. For cancer patients,tumor size, cell count, or cancer specific markers can be monitored.

In one embodiment, the disclosure provides methods for inhibiting growthof a tumor using the FRβ-binding agents or antibodies described herein.In certain embodiments, the method of inhibiting growth of a tumorcomprises contacting a cell with a FRβ-binding agent (e.g., antibody) invitro. For example, an immortalized cell line or a cancer cell line iscultured in medium to which is added an anti-FRβ antibody or other agentto inhibit tumor growth. In some embodiments, tumor cells are isolatedfrom a patient sample such as, for example, a tissue biopsy, pleuraleffusion, or blood sample and cultured in medium to which is added aRSPO-binding agent to inhibit tumor growth.

In some embodiments, the method of inhibiting growth of a tumorcomprises contacting the tumor or tumor cells with a RSPO-binding agent(e.g., antibody) in vivo.

In certain embodiments, the method of inhibiting growth of a tumorcomprises administering to a subject a therapeutically effective amountof a FRβ-binding agent. In certain embodiments, the subject is a human.In certain embodiments, the subject has a tumor or has had a tumor whichwas removed. In some embodiments, the subject has a tumor with anelevated expression level of FRβ. In some embodiments, the FRβ-bindingagent is an antibody. In some embodiments, the FRβ-binding agent isantibody AS04498.

In certain embodiments, the tumor has elevated expression levels of FRβor over-expresses FRβ. In some embodiments, the tumor has a highexpression level of FRβ. In general, the phrase “a tumor has elevatedexpression levels of” a protein (or similar phrases) refers toexpression levels of a protein in a tumor as compared to expressionlevels of the same protein in normal tissue of the same tissue type.However, in some embodiments, the expression levels of a protein in atumor are “elevated” or “high” as compared to the average expressionlevel of the protein within a group of tissue types. In someembodiments, the expression levels of a protein in a tumor are“elevated” or “high” as compared to the expression level of the proteinin other tumors of the same tissue type or a different tissue type.

In certain embodiments, the disclosure provides a method of inhibitinggrowth of a tumor in a subject, comprising administering atherapeutically effective amount of a FRβ-binding agent to the subject.In certain embodiments, the tumor comprises cancer stem cells. Incertain embodiments, the frequency of cancer stem cells in the tumor isreduced by administration of the FRβ-binding agent. In some embodiments,the disclosure also provides a method of reducing the frequency ofcancer stem cells in a tumor, comprising contacting the tumor with aneffective amount of a FRβ-binding agent (e.g., an anti-FRβantibody).

In some embodiments, the tumor is a solid tumor. In certain embodiments,the tumor is a tumor selected from the group consisting of colorectaltumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breasttumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma,cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. Insome embodiments, the tumor is a colorectal tumor that comprises aninactivating mutation in the APC gene. In some embodiments, the tumor isa colorectal tumor that does not comprise an inactivating mutation inthe APC gene.

In certain embodiments, the disclosure provides methods for treatingcancer comprising administering a therapeutically effective amount of aFRβ-binding agent to a subject. In certain embodiments, the cancer ischaracterized by cells expressing elevated levels of FRβ protein ascompared to expression levels of FRβ protein in normal tissue. Incertain embodiments, the cancer is characterized by cellsover-expressing FRβ.

In certain embodiments, in addition to administering a FRβ-binding agent(e.g., an antibody), the method or treatment further comprisesadministering at least one additional therapeutic agent. An additionaltherapeutic agent can be administered prior to, concurrently with,and/or subsequently to, administration of the FRβ-binding agent.Pharmaceutical compositions comprising a FRβ-binding agent and theadditional therapeutic agent(s) are also provided. In some embodiments,the at least one additional therapeutic agent comprises 1, 2, 3, or moreadditional therapeutic agents.

Combination therapy with two or more therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects and/or increasing the therapeuticindex of the agent(s). Combination therapy may decrease the likelihoodthat resistant cancer cells will develop. In some embodiments,combination therapy comprises a therapeutic agent that affects (e.g.,inhibits or kills) non-tumorigenic cells and a therapeutic agent thataffects (e.g., inhibits or kills) tumorigenic CSCs.

In some embodiments, the combination of a FRβ-binding agent and at leastone additional therapeutic agent results in additive or synergisticresults. In some embodiments, the combination therapy results in anincrease in the therapeutic index of the FRβ-binding agent. In someembodiments, the combination therapy results in an increase in thetherapeutic index of the additional agent(s). In some embodiments, thecombination therapy results in a decrease in the toxicity and/or sideeffects of the FRβ-binding agent. In some embodiments, the combinationtherapy results in a decrease in the toxicity and/or side effects of theadditional agent(s).

Useful classes of therapeutic agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascisplatin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, purine antimetabolites, puromycins, radiation sensitizers,steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or thelike. In certain embodiments, the second therapeutic agent is analkylating agent, an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor. In some embodiments, the secondtherapeutic agent is a platinum complex such as carboplatin orcisplatin. In some embodiments, the additional therapeutic agent is aplatinum complex in combination with a taxane.

Therapeutic agents that may be administered in combination with theRSPO-binding agents include chemotherapeutic agents, representative,non-limiting examples of chemotherapeutic agents are recited above.Thus, in some embodiments, the method or treatment involves theadministration of a FRβ-binding agent or antibody disclosed herein incombination with a chemotherapeutic agent or cocktail of multipledifferent chemotherapeutic agents.

Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously. Preparation and dosing schedules for suchchemotherapeutic agents can be used according to manufacturers'instructions or as determined empirically by the skilled practitioner.Preparation and dosing schedules for such chemotherapy are alsodescribed in. The Chemotherapy Source Book, 4^(th) Edition, 2008, M. C.Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Combined administration can include co-administration, either in asingle pharmaceutical formulation or using separate formulations, orconsecutive administration in either order but generally within a timeperiod such that all active agents can exert their biological activitiessimultaneously.

It will be appreciated that the combination of a FRβ-binding agent andat least one additional therapeutic agent may be administered in anyorder or concurrently. In some embodiments, the FRβ-binding agent willbe administered to patients that have previously undergone treatmentwith a second therapeutic agent. In certain other embodiments, theFRβ-binding agent and a second therapeutic agent will be administeredsubstantially simultaneously or concurrently. For example, a subject maybe given a FRβ-binding agent (e.g., an antibody) while undergoing acourse of treatment with a second therapeutic agent (e.g.,chemotherapy). In certain embodiments, a FRβ-binding agent will beadministered within 1 year of the treatment with a second therapeuticagent. In certain alternative embodiments, a FRβ-binding agent will beadministered within 10, 8, 6, 4, or 2 months of any treatment with asecond therapeutic agent. In certain other embodiments, a FRβ-bindingagent will be administered within 4, 3, 2, or 1 weeks of any treatmentwith a second therapeutic agent. In some embodiments, a FRβ-bindingagent will be administered within 5, 4, 3, 2, or 1 days of any treatmentwith a second therapeutic agent. It will further be appreciated that thetwo (or more) agents or treatments may be administered to the subjectwithin a matter of hours or minutes (i.e., substantiallysimultaneously).

For the treatment of a disease, the appropriate dosage of an FRβ-bindingagent (e.g., an antibody) disclosed herein depends on the type ofdisease to be treated, the severity and course of the disease, theresponsiveness of the disease, whether the FRβ-binding agent or antibodyis administered for therapeutic or preventative purposes, previoustherapy, the patient's clinical history, and so on, all at thediscretion of the treating physician. The FRβ-binding agent or antibodycan be administered one time or over a series of treatments lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved (e.g., reduction in tumorsize). Optimal dosing schedules can be calculated from measurements ofdrug accumulation in the body of the patient and will vary depending onthe relative potency of an individual antibody or agent. Theadministering physician can easily determine optimum dosages, dosingmethodologies, and repetition rates. In certain embodiments, dosage isfrom 0.01 μg to 100 mg/kg of body weight, from 0.1 μg to 100 mg/kg ofbody weight, from 1 μg to 100 mg/kg of body weight, from 1 mg to 100mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10mg to 50 mg/kg of body weight. In certain embodiments, the dosage of theantibody or other FRβ-binding agent is from about 0.1 mg to about 20mg/kg of body weight. In certain embodiments, dosage can be given onceor more daily, weekly, monthly, or yearly. In certain embodiments, theantibody or other FRβ-binding agent is given once every week, once everytwo weeks or once every three weeks.

In some embodiments, a FRβ-binding agent (e.g., an antibody) may beadministered at an initial higher “loading” dose, followed by one ormore lower doses. In some embodiments, the frequency of administrationmay also change. In some embodiments, a dosing regimen may compriseadministering an initial dose, followed by additional doses (or“maintenance” doses) once a week, once every two weeks, once every threeweeks, or once every month. For example, a dosing regimen may compriseadministering an initial loading dose, followed by a weekly maintenancedose of, for example, one-half of the initial dose. Or a dosing regimenmay comprise administering an initial loading dose, followed bymaintenance doses of, for example one-half of the initial dose everyother week. Or a dosing regimen may comprise administering three initialdoses for 3 weeks, followed by maintenance doses of, for example, thesame amount every other week.

As is known to those of skill in the art, administration of anytherapeutic agent may lead to side effects and/or toxicities. In somecases, the side effects and/or toxicities are so severe as to precludeadministration of the particular agent at a therapeutically effectivedose. In some cases, drug therapy must be discontinued, and other agentsmay be tried. However, many agents in the same therapeutic class oftendisplay similar side effects and/or toxicities, meaning that the patienteither has to stop therapy, or if possible, suffer from the unpleasantside effects associated with the therapeutic agent.

Thus, the disclosure provides methods of treating cancer in a subjectcomprising using an intermittent dosing strategy for administering oneor more agents, which may reduce side effects and/or toxicitiesassociated with administration of a FRβ-binding agent, chemotherapeuticagent, etc. In some embodiments, a method for treating cancer in a humansubject comprises administering to the subject a therapeuticallyeffective dose of a FRβ-binding agent in combination with atherapeutically effective dose of a chemotherapeutic agent, wherein oneor both of the agents are administered according to an intermittentdosing strategy. In some embodiments, the intermittent dosing strategycomprises administering an initial dose of a FRβ-binding agent to thesubject, and administering subsequent doses of the FRβ-binding agentabout once every 2 weeks.

In some embodiments, the intermittent dosing strategy comprisesadministering an initial dose of a FRβ-binding agent to the subject, andadministering subsequent doses of the FRβ-binding agent about once every3 weeks. In some embodiments, the intermittent dosing strategy comprisesadministering an initial dose of a FRβ-binding agent to the subject, andadministering subsequent doses of the FRβ-binding agent about once every4 weeks. In some embodiments, the FRβ-binding agent is administeredusing an intermittent dosing strategy and the chemotherapeutic agent isadministered weekly.

In certain embodiments, a FRβ binding agent, such as a human anti-humanFRβ mAb or antibody fragments described herein (with or without linkedmoieties) may be administered by any available route including, but notlimited to, oral or parenteral routes of administration such asintravenous, intramuscular, intraperitoneal, subcutaneous, intrathecal,intraarterial, nasal, transdermal (e.g., as a patch), or pulmonaryabsorption. Antibodies or antibody fragments may include a deliveryagent (e.g., a cationic polymer, peptide molecular transporter,surfactant, etc.) as a composition containing a pharmaceuticallyacceptable carrier. As used herein the term “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifugal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into pharmaceuticalformulations as described herein.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Solutions or suspensions used forparenteral administration can include the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,CREMOPHOR® EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline(PBS). In all cases, the composition should be sterile and should befluid to the extent that easy syringability exists. Pharmaceuticalformulations are ideally stable under the conditions of manufacture andstorage and should be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. In general, the relevantcarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be advantageous to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theantibody or antibody fragment in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the purified antibody or antibody fragmentinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,exemplary methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, theantibody or antibody fragment can be incorporated with excipients andused in the form of tablets, troches, or capsules, e.g., gelatincapsules. Oral compositions can also be prepared using a fluid carrierfor use as a mouthwash. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition.The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the a FRβ-binding agent, such as ahuman anti-human FRβ mAb or antibody fragment thereof and a deliveryagent are preferably delivered in the form of an aerosol spray from apressured container or dispenser which contains a suitable propellant,e.g., a gas such as carbon dioxide, or a nebulizer. The presentdisclosure particularly contemplates delivery of the compositions usinga nasal spray, inhaler, or other direct delivery to the upper and/orlower airway. According to certain embodiments, a FRβ-binding agent,such as a human anti-human FRβ mAb or antibody fragment thereof and adelivery agent are formulated as large porous particles for aerosoladministration.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the purified polypeptide or protein anddelivery agents are formulated into ointments, salves, gels, or creamsas generally known in the art.

In certain embodiments, compositions are prepared with carriers thatwill protect the antibody or antibody fragment against rapid eliminationfrom the body, such as a controlled release formulation, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. These can be prepared according to methods known to thoseskilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active antibody or antibodyfragment calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

In certain embodiments, the a FRβ-binding agent, such as a humananti-human FRβ mAb or antibody fragment can be administered at variousintervals and over different periods of time as required. Those ofordinary skill in the art will appreciate that certain factors caninfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Generally, treatment of a subject with aFRβ-binding agent, such as a human anti-human FRβ mAb or antibodyantigen-binding fragment as described herein can include a singletreatment or, in many cases, can include a series of treatments. It isfurthermore understood that appropriate doses may depend upon thepotency of the antibody or antibody fragment and may optionally betailored to the particular recipient, for example, throughadministration of increasing doses until a preselected desired responseis achieved. It is understood that the specific dose level for anyparticular animal subject may depend upon a variety of factors includingthe activity of the specific polypeptide or protein employed, the age,body weight, general health, gender, and diet of the subject, the timeof administration, the route of administration, the rate of excretion,any drug combination, and the degree of expression or activity to bemodulated.

Pharmaceutical formulations as described herein can be included in acontainer, pack, or dispenser together with instructions foradministration.

In some embodiments, an antibody or antigen binding fragment thereof isadministered in combination with one or more pharmaceutical agents(e.g., an anti-inflammatory agent) or antibodies.

V. Kits Comprising FRβ-Binding Agents

The disclosure provides kits that comprise the FRβ-binding agents (e.g.,antibodies) described herein and that can be used to perform the methodsdescribed herein. In certain embodiments, a kit comprises at least onepurified antibody against FRβ protein in one or more containers. In someembodiments, the kits contain all of the components necessary and/orsufficient to perform a detection assay, including all controls,directions for performing assays, and any necessary software foranalysis and presentation of results. One skilled in the art willreadily recognize that the disclosed FRβ-binding agents can be readilyincorporated into one of the established kit formats that are well knownin the art.

Further provided are kits comprising a FRβ-binding agent (e.g., ananti-FRβ antibody), as well as at least one additional therapeuticagent. In certain embodiments, the second (or more) therapeutic agent isa chemotherapeutic agent. In certain embodiments, the second (or more)therapeutic agent is an angiogenesis inhibitor.

The disclosure is further described by the following paragraphs:

1. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising: a heavy chain variable region (V_(H))CDR 1 comprising the amino acid sequence set forth in SEQ ID NO:1; aV_(L) CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2;a V_(H) CDR3 comprising the amino acid sequence set forth in SEQ IDNO:3; a light chain variable region (V_(L)) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO:4; a V_(L) CDR2 comprising theamino acid sequence set forth in SEQ ID NO:5; and a V_(L) CDR3comprising the amino acid sequence set forth in SEQ ID NO:6.2. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising a heavy chain and a light chain, whereinthe heavy chain variable region (V_(H)) CDR 1 has the amino acidsequence set forth in SEQ ID NO:1; a V_(L) CDR2 has the amino acidsequence set forth in SEQ ID NO:2; and a V_(L) CDR3 has the amino acidsequence set forth in SEQ ID NO:3.3. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising a heavy chain and a light chain, whereinthe light chain variable region (V_(L)) CDR1 has the amino acid sequenceset forth in SEQ ID NO: 4; a V_(L) CDR2 has the amino acid sequence setforth in SEQ ID NO: 5; and a V_(L) CDR3 has the amino acid sequence setforth in SEQ ID NO:6.4. The antibody or fragment of an antibody disclosed herein, wherein theantibody or fragment has one or more properties selected from the groupconsisting of: (a) the antibody or fragment does not detectably bind tohuman folate receptor alpha (FRα); (b) the antibody or fragment binds tohuman macrophages but not to mouse macrophages; (c) the antibody orfragment has a binding affinity from 1 to 5 nM; and (d) the antibodymediates antibody-dependent cellular cytotoxicity (ADCC) of FR-βexpressing target cells.5. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ) andhas an equilibrium dissociation constant (K_(D)) of less than 2.5 nM.6. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising: a heavy chain sequence of SEQ ID NO. 8and a light chain sequence of SEQ ID NO. 12.7. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising: a heavy chain sequence of SEQ ID NO. 7and a light chain sequence of SEQ ID NO. 15.8. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising: a heavy chain sequence of SEQ ID NO. 17and a light chain sequence of SEQ ID NO. 20.9. An isolated human monoclonal antibody, or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising: a heavy chain sequence selected fromthe group consisting of SEQ ID NO. 7, 8, 9, 17, 18, and 19 and a lightchain sequence selected from the group consisting of SEQ ID NO. 11, 12,15, 16, 20, and 21.10. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ) with higher affinity than monoclonal antibody m909.11. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ) comprising a heavy chain sequence of SEQ ID NO. 24 with one ormore mutations recited in Table 4, and a light chain sequence of SEQ IDNO. 25 with one or more mutations recited in Table 4.12. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment binds to cell surface FRβ.13. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody is an IgG1 antibody.14. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment is de-fucosylated.15. The antibody or fragment of any of the preceding paragraphs, whereinthe fragment is a Fab antibody fragment, a F(ab′)₂ fragment, or a singlechain antibody fragment (scFv).16. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment is conjugated with a pharmaceutical agent17. The antibody or fragment of any of the preceding paragraphs, whereinsaid pharmaceutical agent is a chemotherapeutic.18. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment is conjugated to a liposome.19. The antibody or fragment of any of the preceding paragraphs, whereinsaid liposome comprises a pharmaceutical agent.20. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment is linked to a toxin.21. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment thereof is covalently linked to said toxin.22. The antibody or fragment of any of the preceding paragraphs, whereinsaid antibody or fragment is linked to a detectable moiety.23. The antibody or fragment of any of the preceding paragraphs, whereinsaid detectable moiety is selected from the group consisting of afluorescent moiety, a luminescent moiety, a radioactive moiety, a CTcontrast agent, an MM contrast agent, and biotin.24. The antibody or fragment of any of the preceding paragraphs, whereinthe antibody or fragment comprises SEQ ID NO:7.25. The antibody or fragment of any of the preceding paragraphs, whereinthe antibody or fragment comprises SEQ ID NO:8.26. The antibody or fragment of any of the preceding paragraphs, whereinthe antibody or fragment comprises SEQ ID NO:9.27. The antibody or fragment of any of the preceding paragraphs, whereinthe antibody or fragment comprises SEQ ID NO:11.28. The antibody or fragment of any of the preceding paragraphs, whereinthe antibody or fragment comprises SEQ ID NO:12.29. A composition comprising the antibody or fragment of any of thepreceding paragraphs and a pharmaceutically acceptable carrier.30. The antibody an antigen-binding fragment thereof of any of thepreceding paragraphs, wherein said antibody mediates antibody-dependentcellular cytotoxicity.31. A method of treating an inflammatory disorder in a subject,comprising administering to the subject a therapeutically effectiveamount of a monoclonal antibody that specifically binds human FRβ, theantibody or fragment comprising: (a) a heavy chain variable region(V_(H)) CDR 1 comprising the amino acid sequence set forth in SEQ IDNO:1; a V_(H) CDR2 comprising the amino acid sequence set forth in SEQID NO:2; a V_(H) CDR3 comprising the amino acid sequence set forth inSEQ ID NO:3; a light chain variable region (V_(L)) CDR1 comprising theamino acid sequence set forth in SEQ ID NO:4; a V_(L) CDR2 comprisingthe amino acid sequence set forth in SEQ ID NO:5; and a V_(L) CDR3comprising the amino acid sequence set forth in SEQ ID NO:6.32. A method of treating cancer in a subject, comprising administeringto the subject a therapeutically effective amount of a monoclonalantibody that specifically binds human FRβ, the antibody or fragmentcomprising: (a) a heavy chain variable region (V_(H)) CDR 1 comprisingthe amino acid sequence set forth in SEQ ID NO:1; a V_(H) CDR2comprising the amino acid sequence set forth in SEQ ID NO:2; a V_(H)CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; alight chain variable region (V_(L)) CDR1 comprising the amino acidsequence set forth in SEQ ID NO:4; a V_(L) CDR2 comprising the aminoacid sequence set forth in SEQ ID NO:5; and a V_(L) CDR3 comprising theamino acid sequence set forth in SEQ ID NO:6.33. A method of reducing the number of FRβ positive cells in a subject,comprising administering to the subject a therapeutically effectiveamount of a monoclonal antibody that specifically binds human FRβ, theantibody or fragment comprising: (a) a heavy chain variable region(V_(H)) CDR 1 comprising the amino acid sequence set forth in SEQ IDNO:1; a V_(H) CDR2 comprising the amino acid sequence set forth in SEQID NO:2; a V_(H) CDR3 comprising the amino acid sequence set forth inSEQ ID NO:3; a light chain variable region (V_(L)) CDR1 comprising theamino acid sequence set forth in SEQ ID NO:4; a V_(L) CDR2 comprisingthe amino acid sequence set forth in SEQ ID NO:5; and a V_(L) CDR3comprising the amino acid sequence set forth in SEQ ID NO:6.34. A method of reducing the number of FRβ positive macrophages in asubject, comprising administering to the subject a therapeuticallyeffective amount of a monoclonal antibody that specifically binds humanFRβ, the antibody or fragment comprising: (a) a heavy chain variableregion (V_(H)) CDR 1 comprising the amino acid sequence set forth in SEQID NO:1; a V_(H) CDR2 comprising the amino acid sequence set forth inSEQ ID NO:2; a V_(H) CDR3 comprising the amino acid sequence set forthin SEQ ID NO:3; a light chain variable region (V_(L)) CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO:4; a V_(L) CDR2comprising the amino acid sequence set forth in SEQ ID NO:5; and a V_(L)CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.35. The method of any of the preceding paragraphs, wherein the antibodyis a recombinant antibody, a chimeric antibody, a bispecific antibody, ahumanized antibody, an IgG1 antibody, an IgG2 antibody, or an antibodyfragment comprising an antigen-binding site.36. The method of any of the preceding paragraphs, wherein the antibodyis a humanized antibody.37. The method of any of the preceding paragraphs, which comprisesadministering at least one additional therapeutic agent.38. The method of any of the preceding paragraphs, wherein theadditional therapeutic agent is a chemotherapeutic agent.39. The method of any of the preceding paragraphs, wherein theadditional therapeutic agent is a second antibody.40. The method of any of the preceding paragraphs, wherein the cancer isselected from the group consisting of acute myeloid leukemia, colorectalcancer, ovarian cancer, pancreatic cancer, leukemia, lung cancer, livercancer, breast cancer, kidney cancer, prostate cancer, gastrointestinalcancer, melanoma, cervical cancer, bladder cancer, glioblastoma, andhead and neck cancer.41. The method of any of the preceding paragraphs, wherein the cancerhas an elevated level of FRβ expression compared to the level of FRβexpression in a corresponding normal tissue.42. The method of any of the preceding paragraphs, further comprising astep of determining the level of FRβ expression in the cancer prior toadministration of the antibody.43. A method of inhibiting growth of a tumor, comprising contacting thetumor with an effective amount of a monoclonal antibody thatspecifically binds human FRβ, the antibody or fragment comprising: (a) aheavy chain variable region (V_(H)) CDR 1 comprising the amino acidsequence set forth in SEQ ID NO:1; a V_(H) CDR2 comprising the aminoacid sequence set forth in SEQ ID NO:2; a V_(H) CDR3 comprising theamino acid sequence set forth in SEQ ID NO:3; a light chain variableregion (V_(L)) CDR1 comprising the amino acid sequence set forth in SEQID NO:4; a V_(L) CDR2 comprising the amino acid sequence set forth inSEQ ID NO:5; and a V_(L) CDR3 comprising the amino acid sequence setforth in SEQ ID NO:6.44. A method of treating an inflammatory disorder comprisingadministering to a subject the antibody or fragment thereof described inany one of the preceding paragraphs.45. A method of treating cancer comprising administering to a subjectthe antibody or fragment thereof described in any one of the precedingparagraphs.46. A method of reducing the number of FRβ positive cells in a subjectcomprising administering to the subject the antibody or fragment thereofdescribed in any one of the preceding paragraphs.47. A method of reducing the number of FRβ positive macrophages in asubject comprising administering to the subject the antibody or fragmentthereof described in any one of the preceding paragraphs.48. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ), the antibody or fragment comprising: a heavy chain sequence ofSEQ ID NO. 7 and a light chain sequence of SEQ ID NO. 11.49. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ), the antibody or fragment comprising: a heavy chain sequence ofSEQ ID NO. 8 and a light chain sequence of SEQ ID NO. 11.50. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ), the antibody or fragment comprising: a heavy chain sequence ofSEQ ID NO. 9 and a light chain sequence of SEQ ID NO. 11.51. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ), the antibody or fragment comprising: a heavy chain sequence ofSEQ ID NO. 7 and a light chain sequence of SEQ ID NO. 12.52. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ), the antibody or fragment comprising: a heavy chain sequence ofSEQ ID NO. 8 and a light chain sequence of SEQ ID NO. 12.53. An isolated human monoclonal antibody, or an antigen-bindingfragment thereof, that specifically binds human folate receptor beta(FRβ), the antibody or fragment comprising: a heavy chain sequence ofSEQ ID NO. 9 and a light chain sequence of SEQ ID NO. 12.54. A chimeric antigen receptor comprising: a target element, whereinthe target element is an antibody or fragment thereof in any one of thepreceding paragraphs, a transmembrane domain, a spacer, a costimulatorydomain, and a signaling domain.55. A fully human, man-made antibody or an antigen-binding fragmentthereof, that specifically binds human folate receptor beta (FRβ), theantibody or fragment comprising: a heavy chain variable region (V_(H))CDR 1 comprising the amino acid sequence set forth in SEQ ID NO:1; aV_(H) CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2;a V_(H) CDR3 comprising the amino acid sequence set forth in SEQ IDNO:3; a light chain variable region (V_(L)) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO:4; a V_(L) CDR2 comprising theamino acid sequence set forth in SEQ ID NO:5; and a V_(L) CDR3comprising the amino acid sequence set forth in SEQ ID NO:6, wherein theantibody does not exist in nature.

Embodiments of the disclosure can be further defined by reference to thefollowing non-limiting examples, which describe in detail preparation ofcertain antibodies of the disclosure and methods for using FRβ-bindingagents or antibodies of the disclosure. It will be apparent to thoseskilled in the art that many modifications, both to materials andmethods, may be practiced without departing from the scope of thepresent disclosure.

EXAMPLES Example 1: Expression of Recombinant FRβ

A nucleic acid encoding a fragment of the human folate receptor beta(FRβ) spanning amino acids 22 to 236 (numbering based on sequence inGenBank Accession No. NP_001107007) was cloned from pcDNA3 tobaculovirus transfer vector pAcGP67 via Sma I and EcoRl sites. FIG. 1contains the amino acid sequence (SEQ ID NO:22) of the FRβ protein.Transfer vector pAcGP67 (BD Biosciences Pharmingen) was co-transfectedwith BaculoGold™ (BD Biosciences Pharmingen) viral DNA into SF9 insectcells according to the manufacturer's instruction.

Recombinant FRβ protein produced from pAcGP67 had four extra residues(ADPG, SEQ ID NO:23) on the N-terminus and five extra histidine residueson the C-terminus. The recombinant FRβ protein was purified fromconditioned medium with a nickel-chelating column, and further purifiedwith a SUPERDEX®75 10/300GL gel filtration column in phosphate bufferedsaline (PBS). The purified protein was >95% purity as estimated bySDS-PAGE. The recombinant FRβ retained its function of binding tofolate.

Example 2: Antibody Selection by Affinity Maturation

A. Paratope Mapping by Alanine Scanning

To determine the contribution of a specific residue to antibody affinityand expression, paratope mapping was performed by alanine scanningmethod. In brief, the variable domains of heavy chain (V_(H)) of M909antibody (FIG. 2A, SEQ ID NO. 24) and light chain (V_(L)) of M909antibody (FIG. 2B, SEQ ID NO. 25) were searched by using NCBI Ig-Blast(http.//www.ncbi.nlm.nih.gov/projects/fgblast/) and complementaritydetermining regions (CDRs) were defined by KABAT delineation system. Allresidues within CDRs were replaced one by one with alanine, or convertedto serine if alanine already existed in the sequences, by site-directedmutagenesis. Then all these mutants (65 in total) were expressed by E.coli in 96-deep-well plates. The crude protein secreted in medium wasanalyzed by ELISA for assessment of antigen-antibody binding affinity.

The key residues are summarized in Table 1. Reference to the key residueis in regard to the m909 sequence (FIGS. 2A and 2B, SEQ ID NOS. 24 and25).

TABLE 1 Key residues identified by paratope mapping Location SignificantModerate CDR-H1 (FIG. 2A) Y27, Y32 F29, A33, H35 CDR-H2 (FIG. 2A) N52I51,G54, N55 CDR-H3 (FIG. 2A) D99, I100, F105, D106 Y102, G103 CDR-L1(FIG. 2B) L27, N33 CDR-L2 (FIG. 2B) G49, Q50, N52 CDR-L3 (FIG. 2B) N95,V97

B. Design and Construction of NNK Libraries

Based on the result of paratope mapping, totally 28 residues wereselected for antibody optimization as shown in Table 2. Residuedesignations for the heavy chain in Tables 2, 3, and 4 correspond to them909 sequence in FIG. 2A (SEQ ID NO. 24) and residues for the lightchain correspond to the m909 sequence in FIG. 2B (SEQ ID NO. 25).Mutants were expressed in E. coli in 96-well plates. The crude proteinsecreted in medium was analyzed by ELISA for assessment ofantigen-antibody binding affinity. The “beneficial mutants” thatincreased antibody affinity were confirmed by affinity ranking. Cloneswith improved dissociation rates were selected for sequencing.

TABLE 2 Residues selected for NNK Library Screening No. VL Region 1 N512 R53 3 P54 4 S55 5 D88 6 S89 7 R90 8 V91 9 S92 10 T93 11 G94 12 N95 13H96 14 V97 15 V98 16 G26 17 T28 18 S31 19 M34 20 W50 21 A53 22 G56 23N57 24 S101 25 Y102 26 G103 27 S104 28 Y107

TABLE 3 Beneficial mutants identified by NNK library screening RegionMutation VL N51F VL R53L VL N95I VH S31Y, S31I VH N57F

C. Design and Construction of Combinatorial Library

Once the “beneficial mutants” were identified, a combinatorial librarywas constructed with random combinations of these mutations. Hundreds ofclones were randomly selected and analyzed by ELISA. The topcombinations of “beneficial mutants” that resulted in highest antibodyaffinity increases were finally selected for affinity ranking. Cloneswith improved dissociation rates were sequenced.

TABLE 4 Affinity matured antibodies identified by combinatorial libraryscreening Clone Identifier VL VH A N51F R53L N95I S31Y N57F B N51F R53LN95I A16V S31Y N57F C N51F N95I S31Y N57F D N51F R53L N95I S31Y E N51FN95I S31Y (ASO4498) F N51F R53L S31Y G N51F R53L N95I S31I H N51F N95IS31I I R53L N95I S31I J N95I S31Y N57F K N95I S31Y L N51F S31I M N51FR53L S31I N N95I S31Y O R53L S31Y P S31I N57F

D. ELISA

Microtiter ELISA plates were coated with 1 μg/ml antigen protein in 100μl coating buffer at 4° C. overnight, and subsequently incubated withblocking buffer at 37° C. for 2 hours. Then the plates were washed withwashing buffer and incubated with 100 μl antibody-containingsupernatants at 37° C. for 1.5 hours. Next the plates were washed withwashing buffer and incubated with 100 μl secondary antibody (0.1 μg/mlgoat antihuman IgG [HRP]) for 45 minutes. After washing, the reactionwas developed with 100 μTMB substrate for 10 minutes at room temperatureand stopped by adding 100 μl of 1 M HCl. The absorbance values weremeasured at 450 nm using a spectrometer.

E. IgG Construction, Expression and Purification

The variable domains of heavy chain (V_(H)) and light chain (V_(L))encoding the M909 and its affinity-matured antibody were synthesized andinserted into pTT5 vector to construct full length IgG expressingvectors, respectively. The heavy and light chain expressing plasmidswere used to co-transfect CHO cells. The recombinant IgGs secreted tothe medium were purified using protein A affinity chromatography.Finally, the concentration and purity of proteins were assessed by OD280and SD S-PAGE, respectively.

Example 3: Binding Characterization of One Representative Antibody

To begin the analysis of the affinity matured antibodies, AS04498 wasselected for further analysis. Antibody affinity to antigen protein wasdetermined using a Surface Plasmon Resonance (SPR) biosensor, BiacoreT200 (GE Healthcare). Antibody was immobilized on the sensor chipthrough Fc capture method. Antigen protein was used as the analyte. Thedata of dissociation (kd) and association (k0) rate constants wereobtained using Biacore T200 evaluation software. The equilibriumdissociation constants (K_(D)) were calculated from the ratio of kd overk0.

FIG. 6A is a line graph displaying the binding properties of chimericIgG to FRβ at various concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25nM, 50 nM, 100 nM, 200 nM, 400 nM.

FIG. 6B is a line graph displaying the binding properties of m909 to FRβat various concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM,100 nM, 200 nM, 400 nM.

FIG. 6C is a line graph displaying the binding properties of AS04498 toFRβ at various concentrations of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50nM (×2), 100 nM, 200 nM, 400 nM.

TABLE 5 provides a summary of the binding data displayed in FIGS. 6A-6C.Rmax Chi² Ligand Analyte k_(a)(1/Ms) K_(d) (1/s) K_(D)(M) (RU) (RU²)Fold Increase Chimeric IgG hFRβ 6.35E+04 0.005986 9.43E−08 121.3 0.263 1 M909 IgG hFRβ 6.41E+04 0.005916 9.24E−08 129.8 0.284 NA ASO4498 hFRβ5.77E+04 1.44E+04 2.50E−09 129.4 0.336 38

As shown in FIGS. 6A-6C and summarized in Table 5, the equilibriumdissociation constant (K_(D)) of AS04498 antibody was at 2.5 nM, 38times higher than the chimeric IgG control. In this particularexperiment, the equilibrium dissociation constant (K_(D)) of m909 was 92nM.

The smaller the dissociation constant, the more tightly bound the ligandis, or the higher the affinity between antibody and receptor. Forexample, an antibody with a nanomolar (nM) dissociation constant bindsmore tightly to a particular receptor than an antibody with a micromolar(μM) dissociation constant.

Example 4: Therapeutic Efficacy of One Representative Antibody

Affinity matured antibody AS04498 binds with higher affinity to FRβcompared to m909. We investigated the efficacy of AS04498 in tumorcontrol as compared to m909.

Materials and Methods

NCr nude mice (athymic nude) were purchased from Taconic BioSciences,Inc (Hudson, N.Y.). Mice were fed Teklad irradiated (sterilized) mousediet and bedded with Teklad irradiated (sterilized) corncob bedding fromEnvigo (Indianapolis, Ind.). Mice were housed in Optimice carouselsterile quarters with filtered air supply in disposable cages fromAnimal Care Systems, Inc. (Centennial, CO).

Cells, 5×10⁶ MV 411 cells in Matrigel, were subcutaneously injected intothe left hind flank of 5-6 week-old athymic nude mice. The study beganwhen tumors reached a mean volume of 192 mm³.

Intraperitoneal dosing occurred on Monday, Wednesday, and Friday with100 μL of study agent or control. The treatment vehicle was PBS. Dosingcontinued for 3 weeks, followed by a three-day observation period. Tumorvolume was measured [(L×W²)/2] by caliper twice per week.

Weight measurements were taken during tumor measurement.Mortality/morbidity was observed and recorded throughout the study. Themice were sacrificed by CO₂ asphyxiation followed by cervicaldislocation at end of study (animals were sacrificed before studycompletion if tumors reached >3,000 mm³).

Results

No adverse events were noted throughout the course of the study. Asdepicted in FIG. 7, administration of 50 μg AS04498 had the greatestanti-tumor effect, which reduced the volume roughly in half. AS04498significantly reduced tumor growth in comparison to m909, and functionedas an effective therapeutic.

Example 5: Antibody Affinity Assay

An antibody affinity assay was performed to compare AS04498 to the m909antibody. Human monocytic leukaemia cell line, THP-1, which was FR-βpositive, was used to determine the dissociation of the antibody fromFR-β expressing cells.

Material and Methods

Cells (100,000 cells) were stained with m909-biotinylated, 2 μl (1μg/ml), AS04498-biotinylated, 2 μl (1 μg/ml), human IgG Isotype, 2 μl (1μg/ml) for 1 hour at 4° C. Cells were washed twice with FACS buffer byadding 500 μl of FACS buffer and spinning down cells at 1200 rpm for 5min.

Non-biotinylated antibodies (m909, AS04498) were added to respectivewells (2 μl (1 μg/ml)) and incubated at 4° C. and 37° C. for differenttime points (0 hour, 2 hour, and 4 hour). Samples were washed at theirrespective time points and stained with SA APC for 20 minutes. Sampleswere washed and analyzed by flow cytometry.

Results

Flow cytometry graphs for binding of m909 antibody and AS04498 are shownin FIGS. 8 and 9. FIG. 8A shows the binding of m909 antibody and AS04498at 4° C. at 0 hour. FIG. 8B shows the binding of m909 antibody andAS04498 at 4° C. at 2 hour time point. FIG. 8C shows the binding of m909antibody and AS04498 at 4° C. at 4 hour time point.

FIG. 9A shows the binding of m909 antibody and AS04498 at 37° C. at 0hour. FIG. 9B shows the binding of m909 antibody and AS04498 at 37° C.at 2 hour time point. FIG. 9C shows the binding of m909 antibody andAS04498 at 37° C. at 4 hour time point

FIG. 10 is a bar graph depicting the binding of the m909 antibody andthe AS04498 antibody. At each time point measured, AS04498 has increasedbinding as comparted to m909. AS04498 has previously been shown to havea higher binding affinity as compared to m909. The data indicates thatAS04498 may remain bound for longer periods of time to FRβ as comparedto the m909 antibody.

At the 2 hour time point, AS04498 has increased binding as compared tothe m909 antibody. This indicates that AS0449 may have a slower off ratethan m909.

Example 6: Mv-411 Xenograft Model

We compared the m909 antibody and the AS04498 antibody in a mousexenograft model.

Material and Methods

MV-411 cells in Matrigel (5×10⁶) were injected subcutaneously into lefthind flank of 5-6 week old athymic nude mice on Day 0. There were eightmice per group.

Dosing was initiated (day 0) when tumors reached a mean volume of 250mm³. Study agent (100 μL) or vehicle (PBS) was administeredintraperitoneal (IP) on Monday, Wednesday, and Friday for 3 weeks. Afterdosing ended, mice were observed for an additional 3 weeks. Tumor volume[(L×W²)/2] was measured by caliper twice per week.

Results

As depicted in FIG. 11, AS04498 inhibited tumor growth more effectivelythan the m909 antibody. AS04498 performed more effectively at multipletime points, and the disparity in effectiveness of the two antibodiesincreased with increasing time periods. Not to be bound by anyparticular theory, AS04498 has a higher affinity for FR-0, and increasedtherapeutic efficacy.

Example 7: Apoptosis Assay Materials and Methods

CHOb and CHO-K1 cells were plated at 0.3⁶ cells/ml in a 12 well plate.The plates were incubated at 37° C. incubator for 24 hours. Cells wereharvested and dead cells floating in the supernatant were collected.

Cells were stained by adding Annexin-V antibody in Annexin V bindingbuffer and 7-AAD stain at the same time. An aliquot of CHOb and CHO-K1cells were incubated at 0 and 10 μg/ml of AS04498 (AM), and 10 μg/mlm909 to examine FRβ expression.

After incubation at 4° C., samples were analyzed on Fortessa flowcytometry machine.

Results

10,000 events were collected. Apoptotic events were analyzed at a 12hour time point. As shown in Table 6, higher early apoptotic FRβ+ cellswith m909. Conversely, as shown in Table 7, there were higher lateapoptotic total cells with m909

TABLE 6 Apoptotic events in FR-β Positive Cells Late Early NecroticApoptosis Apoptosis Live cells FR-β + cells q1 q2 q3 q4 CHOb0 15.6%75.6% 5.56% 3.31% CHOb m909 10 10.3% 63.0% 17.1% 9.60% CHOb_m909_APC_1010.5% 57.7% 20.4% 11.5% CHOb AM 10 16.1% 77.0% 4.01% 2.94%CHOb_Am_APC_10 17.0% 77.1% 3.76% 2.18%

TABLE 7 Apoptotic Events in Total Cells Late Early Necrotic ApoptosisApoptosis Live cells Total Cells q1 q2 q3 q4 CHOb HUIgG 0 1.26% 23.4%62.1% 13.2% CHOb0 1.26% 25.8% 60.3% 12.7% CHOb m909 10 2.08% 42.1% 45.8%10.1% CHOb_m909_APC_10 2.42% 47.5% 40.2% 9.92%  CHOb AM 10 1.43% 25.5%60.4% 12.7% CHOb_Am_APC_10 2.07% 27.7% 57.8% 12.4%

Example 8: Mouse Tumor and Blood Analysis

We investigated the ability of FRβ to bind and deplete macrophages inmouse blood and tumor samples.

Materials and Methods

THP-1 cells were administered intravenously on day 0. Dosing wasinitiated on day 2. Mice were dosed by the IP route MWF for a total of10 doses. On day 42, tumors were removed from the mice and digested withGentleMACs.

Blood was collected using cardiac puncture. Cells were isolated andstained for analysis by flow cytometry.

Results

The percentage of macrophages in the mouse blood and tumor samples wasdetermined by the percentage of mCD11b and F4-80 double positive cells(see Table 8 for markers). As shown in FIG. 12A, AS04498 effectivelydepleted or reduced the number of macrophages in the mouse blood sample.AS04498 performed better than the m909 antibody.

As shown in FIG. 12B, AS04498 effectively depleted or reduced the numberof macrophages in the mouse tumor sample. AS04498 performed better thanthe m909 antibody.

TABLE 8 Markers used to identify macrophages Marker Recognize CD33_PETransmembrane receptor expressed on myeloid lineage cells M909_-Biotin,SA-APC FRβ positive cells F4/80_PE-Cy7 Mouse macrophage markermCD11b-Pacific Blue Mouse macrophage marker Aqua Live Dead Live/deadcells GFP THP-1 cells engineered to express GFP-Luc- tumor marker

Example 9: Humanized Thioglycollate Inflammation Model Materials andMethods

Hu-CD34 NSG™ mice that were engrafted with human CD34+ cells with >25%human CD45+ cells in the peripheral blood were used for the study. Micewere at least 12 weeks post engraftment. Attempts were made todistribute donors equally into groups of 5.

The mice were injected IP with 4% thioglycolate on Day 0 and dosed with1 μg or 5 of m909 or AS04498 antibody on Day 2.

All animals were euthanized by CO₂ asphyxiation on Day 4 and cells werecollected via peritoneal lavage. Samples were analyzed for the followingmarkers: hCD45, CD68, CD14, anti-FRb, and viability dye (7AAD). Countingbeads were included in each sample for absolute cell numberdeterminations.

Results

As shown in FIG. 13, AS04498 effectively inhibited cells positive forboth CD68, and FRβ. AS04498 was more effective than m909 in inhibitingcells positive for both CD68 and FRβ positive cells. AS04498 waseffective at both 1 μg and 5 μg doses.

As shown in FIG. 14, AS04498 effectively inhibited CD68 positive cells.In this case, AS04498 was more effective at the 5 μg dose.

As shown in FIG. 15, AS04498 effectively inhibited cells positive forboth CD14 and FRβ. AS04498 was more effective than m909 in inhibitingcells positive for both CD14, and FRβ. AS04498 was effective at both 1μg and 5 μg doses.

As shown in FIG. 16, AS04498 effectively inhibited cells positive forCD14. AS04498 was more effective than m909 in inhibiting cells positivefor CD14. AS04498 was more effective at the 5 μg dose.

As shown in FIG. 17, AS04498 effectively inhibited cells positive forFRβ. AS04498 was more effective than m909 in inhibiting cells positivefor FRβ.

It is to be understood that while the antibodies, fragments thereof,compounds, and methods have been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the disclosure, which is definedby the scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims.

INFORMAL SEQUENCE LISTING SEQ ID NO. 1 (S31Y TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN COMPLEMENTARY DETERMININGREGION FROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY SEQUENCE 1GLY TYR THR PHE THR TYR TYR ALA SEQ ID NO. 2 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN COMPLEMENTARY DETERMININGREGION FROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY SEQUENCE 2LYS TYR SER GLN LYS PHE GLN SEQ ID NO. 3 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN COMPLEMENTARY DETERMININGREGION FROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY SEQUENCE 3ALA ARG ASP ILE SER TYR GLY SER PHE ASP TYR TRP SEQ ID NO. 4 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN COMPLEMENTARY DETERMINING REGIONFROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY SEQUENCE 4SER LEU ARG SER ASN TYR SEQ ID NO. 5 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN COMPLEMENTARY DETERMINING REGIONFROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY (N51F) SEQUENCE 5 GLY GLN PHESEQ ID NO. 6 TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN COMPLEMENTARY DETERMINING REGIONFROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY (N95I) SEQUENCE 6ASP SER ARG VAL SER THR GLY ILE HIS VAL VAL PHE SEQ ID NO. 7 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (S31Y) SEQUENCE 7EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVT VSSSEQ ID NO. 8 TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (S31Y) SEQ ID NO. 8EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP SEQ ID NO. 9TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (S31Y) SEQ ID NO. 9MGWSWILLFLLSVTAGVHSEVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 10 (DNA ENCODING SEQ ID NO. 9) TYPE: DNAORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: NUCLEIC ACID SEQUENCE ENCODING HEAVY CHAINFROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY (S31Y) SEQ ID NO. 10ATGGGCTGGAGCTGGATCCTGCTGTTCCTCCTGAGCGTGACAGCAGGAGTGCACAGCGAGGTGCAGCTGGTCCAGTCCGGCGCTGAGGTGAAGAAACCAGGGGCATCCGTGAAAGTCTCCTGCAAAGCCAGTGGCTACACCTTCACA TAC TATGCCATGCACTGGGTGAGGCAGGCTCCAGGACAGCGACTGGAATGGATGGGCTGGATCAACGCCGGCAACGGGAATACTAAGTACTCCCAGAAATTTCAGGGGCGGGTGACTATTACCAGAGACACCTCAGCCAGCACAGCTTATATGGAGCTGAGCTCCCTGCGAAGCGAAGATACAGCAGTCTACTATTGTGCCAGAGACATTAGTTACGGAAGCTTTGATTATTGGGGACAGGGCACACTGGTGACTGTCTCTAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 11TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY SEQUENCE 11SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLG SEQ ID NO. 12TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY SEQUENCE 12SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO. 13 (DNA ENCODING SEQ ID NO. 12) TYPE: DNAORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: NUCLEIC ACID SEQUENCE ENCODING LIGHT CHAINFROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY SEQUENCE 13ATGGGCTGGAGCTGGATCCTGCTGTTCCTCCTGAGCGTGACAGCAGGAGTGCACAGCTCTTCTGAACTTACTCAAGATCCCGCCGTGTCCGTGGCCCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCAACTACGCCAATTGGTACCAGCAGAAGCCCGGCCAGGCCCCCGTGCTGGTCATCTACGGCCAGTTCAACAGGCCCTCCGGCATCCCCGACCGCTTCTCCGGCTCCTCCTCCGGCAACACCGCCTCCCTGACCATCACCGGCGCCCAGGCCGCAGACGAGGCCGACTACTACTGCGATTCCAGGGTGTCCACCGGCATCCACGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGCTGGGCCAGCCCAAGGCCGCCCCCTCCGTGACCCTGTTCCCCCCCTCCTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCTCCGACTTCTACCCCGGCGCCGTGACCGTGGCCTGGAAGGCCGACTCCTCCCCCGTGAAGGCCGGCGTGGAGACAACCACCCCCTCCAAGCAGTCCAACAACAAATACGCCGCCTCCTCCTACCTGTCCCTGACCCCCGAGCAGTGGAAGTCCCACAGGTCCTACTCCTGCCAAGTCACCCACGAGGGCTCCACCGTGGAGAAGACCGTGGCCCCCACCGAGTGCTCCTAG SEQ ID NO. 14 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN COMPLEMENTARY DETERMINING REGIONFROM A HUMAN ANTI-HUMAN FR BETA ANTIBODY SEQUENCE 14 GLY GLN ASNSEQ ID NO. 15 (N 95 I) TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY SEQUENCE 15SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLG SEQ ID NO. 16 (N951)TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY SEQUENCE 16SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO. 17 TYPE: PRTORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (S31Y; N57F) SEQUENCE 17EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGFTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVT VSSSEQ ID NO. 18 TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (S31Y; N57F) SEQ ID NO. 18EVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGFTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP SEQ ID NO. 19TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: HEAVY CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (S31Y; N57F) SEQ ID NO. 19MGWSWILLFLLSVTAGVHSEVQLVQSGAEVKKPGASVKVSCKASGYTFTYYAMHWVRQAPGQRLEWMGWINAGNGFTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO. 20TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (N51F; R53L; N95I) SEQUENCE 20SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNLPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLG SEQ ID NO. 21TYPE: PRT ORGANISM: ARTIFICIAL SEQUENCEOTHER INFORMATION: LIGHT CHAIN SEQUENCE FROM A HUMAN ANTI-HUMANFR BETA ANTIBODY (N51F; R53L; N95I) SEQUENCE 21SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQFNLPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGIHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO. 22 Type: PRTOrganism: Homo Sapiens (FRβ) SEQ ID NO. 22MVWKWMPLLLLLVCVATMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQSWRKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVARFYAAAMHVNAGEMLHGTGGLLLSLALMLQLWLLG SEQ ID NO. 23 Type: PRTOther information: N-terminal amino acids from recombinantlyproduced FR beta SEQ ID NO. 23 ALA ASP PRO GLY (m909 heavy)SEQ ID NO. 24 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDISYGSFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTS (m909 light)SEQ ID NO. 25SSELTQDPAVSVALGQTVRITCQGDSLRSNYANWYQQKPGQAPVLVIYGQNNRPSGIPDRFSGSSSGNTASLTITGAQAADEADYYCDSRVSTGNHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

What is claimed is:
 1. An isolated human monoclonal antibody, or anantigen-binding fragment thereof, that specifically binds human folatereceptor beta (FRβ), and having an equilibrium dissociation constant(K_(D)) for FRβ of 2.5 nM or less.
 2. The antibody or fragment of claim1, wherein the antibody or fragment has one or more properties selectedfrom the group consisting of: (a) the antibody or fragment does notdetectably bind to human folate receptor alpha (FRα); (b) the antibodyor fragment binds to human macrophages but not to mouse macrophages; and(c) the antibody mediates antibody-dependent cellular cytotoxicity(ADCC) of FR-β expressing target cells.
 3. A composition comprising theantibody or fragment of claim
 1. 4. A chimeric antigen receptorcomprising the antibody or fragment of claim
 1. 5. A method of treatingcancer in a subject, comprising administering to the subject atherapeutically effective amount of a monoclonal antibody thatspecifically binds human FRβ, the antibody or fragment comprising: (a) aheavy chain variable region (V_(H)) CDR 1 comprising the amino acidsequence set forth in SEQ ID NO: 1; a V_(H) CDR2 comprising the aminoacid sequence set forth in SEQ ID NO: 2; a V_(H) CDR3 comprising theamino acid sequence set forth in SEQ ID NO: 3; a light chain variableregion (V_(L)) CDR1 comprising the amino acid sequence set forth in SEQID NO: 4; a V_(L) CDR2 comprising the amino acid sequence set forth inSEQ ID NO: 5; and a V_(L) CDR3 comprising the amino acid sequence setforth in SEQ ID NO:
 6. 6. The method of claim 5, wherein the antibody isa recombinant antibody, a chimeric antibody, a bispecific antibody, ahumanized antibody, an IgG1 antibody, an IgG2 antibody, or an antibodyfragment comprising an antigen-binding site.
 7. The method of claim 5,further comprising administering at least one additional therapeuticagent.
 8. The method of claim 7, wherein the additional therapeuticagent is a chemotherapeutic agent.
 9. The method of claim 7, wherein theadditional therapeutic agent is a second antibody.
 10. The method ofclaim 5, wherein the cancer is selected from the group consisting ofacute myeloid leukemia, colorectal cancer, ovarian cancer, pancreaticcancer, leukemia, lung cancer, liver cancer, breast cancer, kidneycancer, prostate cancer, gastrointestinal cancer, melanoma, cervicalcancer, bladder cancer, glioblastoma, and head and neck cancer.